Method for detecting and identifying the presence of biological materials derived from fish and oligonucleotides therefor

ABSTRACT

A subject of the present invention is a method for detecting the presence of biological materials originating from gadiformes chosen from the group constituted by the gadidae, the merluccidae, the macrouridae and/or the moridae, in a sample of organic material, characterized in that the presence of mitochondrial DNA originating from gadiformes chosen from the group constituted by the gadidae, the merluccidae, the macrouridae and/or the moridae in said organic material is determined by amplification of at least one sequence or fragment of mitochondrial DNA specific to the genome of the gadiformes chosen from the group constituted by the gadidae, the merluccidae, the macrouridae and/or the moridae, and contained in the mitochondrial DNA extracted from said sample, namely at least one sequence or fragment present in the genomes of the gadiformes chosen from the group constituted by the gadidae, the merluccidae, the macrouridae and/or the moridae, but absent from the genomes of the other animal genera, and in particular of the other animal species.

A subject of the present invention is a method for detecting andidentifying the presence of biological materials originating fromgadiformes, in a sample of organic material.

It also has as a subject the oligonucleotides for implementing thismethod, and the DNA fragments obtained using said oligonucleotides.

The increasing economic value of certain animal species used for humanfood is linked to a major imbalance between supply and demand in themarket. A direct consequence of this is the introduction of an increasein the practice of food adulteration. One of the most common ways ofadulterating food comprises the substitution of components originatingfrom animal species of high commercial value by those originating fromspecies of less value, or even by components of plant origin such assoya.

This uncontrolled exploitation of biodiversity is even more pronouncedin the fishing industry, where industrial processes such as theproduction of fillets or on-board freezing make it difficult, evenimpossible, to identify the species that are caught. In addition, thisindustrialization of fishing allows new species of fish to be caught,which are deep-sea fish.

The “interspecific” adulteration of food products is therefore at oneand the same time a problem of economics, of public health and ofpreservation of the environment, which affects equally consumers,distributors, producers and the authorities responsible for food hygieneand fraud prevention.

It is therefore important to be able not only to determine the presencein food of biological materials originating from animal species, butalso to identify the origin of these biological materials present infood.

In the agri-foodstuffs sector, the characterization of animal speciesmakes use of biochemical techniques for the analysis of proteins(electrophoresis and immunoanalysis) or chemical techniques (principallychromatography). Thus, polyacrylamide gel electrophoresis underdenaturing conditions was essentially developed for the analysis ofcooked food (analysis of peptides or proteins denatured and coagulatedby cooking (Patterson R. L. S., 1985, Biochemical identification of meatspecies, Elsevier ed)). This technique is now supplanted by pH gradientelectrophoresis (isoelectric focalization or “IEF”), with much greaterresolution. The fishing industry currently uses this method for theidentification of species of fish (comparison of the electrophoreticprofile of the muscle proteins of the studied species with anelectrophoretic reference profile (Sotelo C. G. et al., 1993, Trends inFood Science and Technology, 4: 395-401)). However, isoelectricfocalization is a technique that is difficult to use.

Although these techniques remain used for the identification of commonspecies (in particular domesticated species) in food, the multiplicationof the number of wild species affected (game, fish, crustaceans,shellfish, molluscs) means that the majority of these techniques are nowat their limits.

Analysis of the genome by means of nucleic probes clearly represents anew alternative for the identification of species, one still littledeveloped at the present time. In fact, the work carried out on ancientDNA (or fossil DNA) since the beginning of 1990s has demonstrated thatDNA is a very stable molecule after death, despite the action of thetime and of the environment (Brown et al., 1994, Bioessays, 16 (10):719-26.). However, when it sur present in small quantities, in the formsof damaged and chemically modified molecules (Pääbo et al., 1989,Journal of Biology Chemistry, 264, 9709-9712). These characteristics aredue essentially to the phenomena of hydrolysis and oxidation (Lindahl,1993, Nature, 362, 709-715). Thanks to the polymerase chain reaction(“PCR” technique) which constitutes a tool of remarkable analyticalpower, it is possible to multiply a given DNA fragment in vitro in analmost exponential manner. By amplifying DNA of a food preparation whichhas undergone modifications such as cooking, salting, smoking, phenomenaof hydrolysis and oxidation, it will be possible to identify eachconstituent of animal or plant origin. PCR was thus recently used forthe characterization of cooked pigmeat (Meyer et al., 1995, Journal ofAOAC International, 78, (6) 1542-1551)), sheep's meat or goat meat(Chikuni et al., 1994, Animal Science and Technology, 65 (6), 571-579)by amplification of sequences specific to the sought species. Similarly,the amplification of sequences specific to the Y chromosome allowed thedetermination of the sex of butchery carcasses of bovine and ovineorigin (Cotinot C. et al., 1991, Genomics. 10 (3): 646-53, Apparao K. B.C., 1995, Meat Science, 39 (1) 123-126).

A subject of international application WO 98/50401 is a method fordetecting the presence of biological materials of bovine origin in asample of organic material. The method described in this reference makesuse of PCR using suitable oligonucleotides, amplifying a part of themitochondrial DNA control region. However, the method described in thisreference allows only the detection of the presence or the absence of asingle bovine species, namely the species Bos taurus, and thus does notallow the detection and identification of the presence of severalspecies.

Fish belonging to the order of the gadiformes constitute the largestgroup of fish that are caught and sold, representing a little more thanhalf of total fish catches. These fish are much used in theagri-foodstuffs sector (fillet, soup, terrine, pates, fish-basedpreparation, oils, flours . . . ) and are therefore very exposed toadulteration. The gadiformes belong to the class of the actinopterygiiand form part of the teleostei or bony fish. They are divided intoseveral families, such as the gadidae (whiting, cod, pollock, ling . . .), the merluccidae (hake), the macrouridae (grenadier), the moridae(mora) and other families that are not industrially exploited.

From a technical point of view, studies have been conducted on themitochondrial DNA (mtDNA) of the gadiformes, which is organized in thesame way as for mammals. mtDNA is an excellent marker of species whichis often used in phylogeny. In fact, depending on the species that isbeing studied, certain portions of mtDNA permit species to bedifferentiated one from the other, others have a finer power ofresolution and allow different populations (geographical races,sub-species) to be distinguished: mtDNA replication control region,regions coding for cytochrome b or coding for the mitochondrial RNAs(RNA 12S or RNA 16S). The majority of the studies carried out on themtDNA of the gadiformes analyse the genetic diversity of the speciesfrom a population point of view, comparing the sequences one withanother or using microsatellite markers (Purcell et al., 1996. MolecularMarine Biology and Biotechnology, 5(3) 185-192; Ruzzante et al., 1998.Molecular Ecology. 7: 1663-1680; Lage and Kornfiels, 1999. MolecularEcology 8: 1355-1357). Other studies are based on a precise region ofthe mtDNA, and calculate rates of divergence in order to study theevolution of one species relative to another (Carr et al., 1999.Canadian Journal of Zoology 77: 19-22). It is possible also to retracetheir evolution and provide fresh data as regards the place of a specieson a phylogenic tree (Morita., 1999. Molecular Phylogeny of Evolution 13(3) 447-454). mtDNA has been sequenced in its entirety in a singlespecies of gadidae: the Atlantic cod (Gadus morhua) (Johansen and Bakke,1996. Molecular Marine Biology and Biotechnology 5(3) 203-214).

A study of the state of the art thus shows that work on the analysis ofDNA, and in particular the mtDNA of certain species of gadiformes, hasalready been carried out. However, no document of the prior artdescribes a method allowing the detection of at least one species offish (in particular of gadiformes) present in a sample of organicorigin, and identification of the species present in said sample, byusing the PCR technique using suitable oligonucleotides. No document ofthe prior art describes a specific, sensitive and reliable method ofdetecting and identifying degraded or non-degraded DNA, originating fromone or more different species of gadiformes, in any sample of organicorigin displaying very varied compositions that can contain fish. Theapplicant therefore endeavoured to develop a sensitive and reliablemethod that allows these shortcomings to be remedied.

One of the aims of the present invention is to provide a method fordetecting the presence of biological materials originating from fish ina sample of organic material.

One of the other aims of the invention is to provide a method foridentifying the genus, in particular of at least one species of fishpresent in a sample of organic material.

Another aim of the invention is to provide a method allowing species offish which, though phylogenetically close, have different commercialvalues, to be distinguished one from the other.

Another aim of the invention is to provide a method of identifying thegenus, in particular of at least one species of fish present in fresh orprocessed food (cooked, lyophilized, dried, pickled, appertized,pasteurized etc.).

A subject of the present invention is a method for detecting thepresence of biological materials originating from gadiformes chosen fromthe group constituted by the gadidae, the merluccidae, the macrouridaeand/or the moridae, in a sample of organic material, characterized inthat the presence of mitochondrial DNA originating from gadiformeschosen from the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, is detected in said organic material byamplification of at least one sequence or fragment of mitochondrial DNAspecific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae, and contained in the mitochondrial DNA extracted from saidsample, namely at least one sequence or fragment present in the genomesof the gadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, but absent from thegenomes of the other animal genera, and in particular of the otheranimal species.

The present invention also has as a subject a method for detecting thepresence of biological materials originating from gadiformes chosen fromthe group constituted by the gadidae, the merluccidae, the macrouridaeand/or the moridae, in a sample of organic material, and for identifyingthe genus, in particular of at least one species of gadiformes chosenfrom the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, present in said sample, characterized inthat the presence of mitochondrial DNA originating from gadiformeschosen from the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, is detected in said organic material byamplification of at least one sequence or fragment of mitochondrial DNAspecific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae, and contained in the mitochondrial DNA extracted from saidsample, namely at least one sequence or fragment present in the genomesof the gadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, but absent from thegenomes of the other animal genera, in particular of the other animalspecies, and in that at least one sequence or fragment of mitochondrialDNA specific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae, thus amplified, is compared with other sequences ofmitochondrial DNA of the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae, said sequence of mitochondrial DNA or said fragment ofmitochondrial DNA specific to the genome of the gadiformes chosen fromthe group constituted by the gadidae, the merluccidae, the macrouridaeand/or the moridae, thus amplified, displaying at least approximately50% identity, in particular approximately 60% identity with the otheraforementioned sequences of mitochondrial DNA of the genome of thegadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae.

By genus is meant an obligatory category to which every species mustbelong and which contains a species or a group of species (Wily, 1981).

By species is meant a population group really or partially capable ofcrossing, and which is reproductively isolated from the other groupshaving the same property. The animal species is divided intosub-species, races, varieties and strains; several neighbouring speciesform a genus, which for its part is a subdivision of the family.

By organic material is meant any solid or liquid material that can bepresumed to have at least partially an organic origin.

The percentage identity relates to the result of the comparison of thenucleic acids of the DNA sequence that it is sought to identify withthose of the known DNA sequences of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae.

Thus, when an amplified DNA sequence or fragment specific to the genomeof the gadiformes displays at least approximately 50% identity, inparticular approximately 60% with the other known DNA sequences of thegenome of the gadiformes, it can be deduced from this that the sample oforganic material to be analysed contains biological materialsoriginating from gadiformes, such as the gadidae, the merluccidae, themacrouridae and/or the moridae.

When an amplified DNA sequence or fragment specific to the genome of thegadiformes displays less than approximately 50% identity with the otherknown DNA sequences of the genome of the gadiformes, it can be deducedfrom this that the sample of organic material to be analysed does notcontain biological materials originating from gadiformes, such as thegadidae, the merluccidae, the macrouridae and/or the moridae.

According to an advantageous embodiment of the invention, the methodallows the detection and optionally the identification of the presenceof gadidae, in particular chosen from the group constituted by Gadusniorhua (common cod), Melanogranimus aeglefinus (haddock), Merlangiusmerlangus (whiting), Micromesistius poutassou (blue whiting), Pollachiusvirens (pollock), Pollachius pollachius (pollack), Trisopterus luscus(common pout), Trisopterus minutus capelanus (poor cod), Theragrachalcogramma (Alaskan pollock), Brosme brosme (tusk), Molva molva (ling)or Molva dypterygia dypterigia (blue ling).

According to another advantageous embodiment of the invention, themethod allows the detection and optionally the identification of thepresence of merluccidae, in particular chosen from the group constitutedby Merluccius albidus (offshore hake), Merluccius australis (Southernhake), Merluccius bilinearis (silver hake), Merluccius capensis(shallow-water Cape hake), Merluccius gayi (Chilean hake), Merlucciushubbsi (Argentine hake), Merluccius merluccius (common hake), Merlucciusparadoxus (deep-water Cape hake), Merluccius productus (North Pacifichake), Merluccius senegalensis (Senegalese hake), Steindachneriaargentea (silver hake).

According to another advantageous embodiment of the invention, themethod allows the detection and optionally the identification of thepresence of macrouridae, in particular chosen from the group constitutedby Abyssicola macrochir (abyssal grenadier), Albatrossia pectoralis(giant grenadier), Bathygadus macrops (grenadier sp.), Gadomus arcuatus(grenadier sp.), Coelorinchus argentatus (silver whiptail grenadier),Coryphaenoides acrolepis (Pacific grenadier), Coryphaenoides mexicanus(Mexican grenadier), Coryphaenoides rupestris (roundnose grenadier),Cynomacrurus pirei (dogtooth grenadier), Hymenocephalus italicus(Italian grenadier), Lepidorhynchus denticulatus (javelin grenadier),Macrourus berglax (grey grenadier), Malacocephalus laevis (beardedgrenadier), Mataeocephalus acipenserinus (sturgeon grenadier), Nezumiaaequalis (smooth grenadier), Sphagemacrurus hirundo (graylinggrenadier), Trachonurus sulcatus (bristly grenadier), Trachyrincushelolepis (armourhead grenadier), Ventrifossa atherodon (arrowtoothgrenadier).

According to another advantageous embodiment of the invention, themethod allows the detection and optionally the identification of thepresence of moridae, such as mora moro (common mora).

Advantageously, each of the amplified sequences or fragments of thegenome of the gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae, is ofmitochondrial origin.

The choice of a mitochondrial sequence is particularly advantageous as,in an animal cell there are approximately 100 to 1000 copies ofmitochondrial DNA for one copy of nuclear DNA. In case of degradation ofthe DNA, the probability of detecting mitochondrial DNA is thereforemuch greater than the probability of detecting nuclear DNA.Mitochondrial DNA can therefore be more surely detected in organicmaterials in which the DNA is subject to various physical (temperature,pressure etc.), chemical or biochemical factors leading to itsdegradation.

According to an advantageous embodiment of the method of the invention,the DNA extracted from the sample of organic material is:

non-degraded DNA originating in particular from a fresh sample or,

degraded DNA, originating in particular from a sample that has beenprocessed, in particular cooked, lyophilized, dried, pickled,appertized, pasteurized etc.

Preferably, the amplification of at least one DNA sequence or fragmentspecific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae, is carried out by the polymerase chain amplification (PCR)method, comprising a repetition of the cycle of the following stages:

heating of the DNA extracted from the sample of organic material, so asto separate the DNA into two single-chain strands,

hybridization of oligonucleotide primers to the monocatenary DNA strandsat an adequate temperature, and

elongation of the oligonucleotide primers by a polymerase at an adequatetemperature, in order to obtain at least one amplified DNA sequence orfragment specific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae.

In the above and in the following, each of the amplified DNA sequencesor fragments obtained at the end of the polymerase chain reaction (PCR)using the primers of the invention, can also be called “amplified DNAfragment”, “DNA fragment”.

By “amplification product” is meant in the above and in the followingthe amplified DNA fragment or fragments or sequence or sequencesobtained at the end of the polymerase chain reaction (PCR). Theamplification product comprises several copies of different amplifiedDNA fragments or sequences when the sample of organic material to beanalysed comprises a mixture of different DNA fragments originating fromdifferent species of gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae. Theamplification product contains several copies of the same amplified DNAfragment or sequence when the sample of organic material to be analysedcomprises several DNA fragments originating from the same species ofgadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae.

In the above and in the following, the oligonucleotide primers can alsobe called “oligonucleotides” or “primers”.

A subject of the invention is also a method for obtaining at least oneDNA sequence or fragment originating from gadiformes, and in particularin gadidae such as those mentioned above, displaying a determined sizeand sequence, specific to the gadiformes, and in particular to thegadidae, from a sample of organic material, a method from which there isamplified, by polymerase chain reaction (PCR), at least one determinedsequence of the genome of the gadiformes, and in particular of thegadidae, present in the genomes of the gadiformes, and in particular ofthe gadidae, but absent from the genomes of the other animal species.

A subject of the invention is also a method for obtaining at least oneDNA sequence or fragment originating from gadiformes, and in particularin merluccidae such as those cited above, displaying a determined sizeand sequence, specific to the gadiformes, and in particular to themerluccidae, from a sample of organic material, a method from whichthere is amplified, by polymerase chain reaction (PCR), at least onedetermined sequence of the genome of the fishs, in particular of thegadiformes, and in particular of the merluccidae, present in the genomesof the gadiformes, and in particular of the merluccidae, but absent fromthe genomes of the other animal species.

In the above and in the following, the expression <<at least one DNAsequence or fragment>> means either that there are several copies of thesame DNA fragment or sequence, or that there are several copies ofdifferent DNA fragments or sequences.

According to an advantageous embodiment of the method of the invention,each of the amplified sequences or fragments of the genome of thegadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, is situated in thecentral part of the gene coding for the cytochrome c oxidase of themitochondrial DNA, delimited by the nucleotides situated in the vicinityof positions 6100 and 6601, and in particular in the vicinity ofpositions 6120 and 6590, and preferably in the vicinity of positions6131 and 6580 of the gene coding for the cytochrome c oxidase of themitochondrial DNA, said positions being defined according to Johansenand Bakke, 1996, Molecular Marine Biology and Biotechnology, 5(3)203-214.

A subject of the invention is also the oligonucleotides chosen fromthose:

(1)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)1 (positions 6131 to6154 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TRT AYC ARC AYY TRT TYT GRT TCT K

in which R is A or G, Y is C or T, K is G or T,

on condition that the following sequences are excluded: CGG GAT CCT GTTCTG ATT CTT GAT TTC C and CGA CGG GAT CCC AAC ACC TGT TTC GAT CAT CGCGGC AAC

or comprising the following sequence SEQ ID N^(o)2 (positions 6134 to6154 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): AYC ARC AYY TRT TYT GRT TCT

in which Y is C or T, R is A or G,

or constituted by the following sequence SEQ ID N^(o)3 (positions 6139to 6153 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): AYY TRT TYT GRT TCT

in which Y is C or T, R is A or G,

or those

(2)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)4 (positions 6244 to6269 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TTY GGN YAY ATR GGN ATR GTN TGA GC

in which Y is C or T, N is A, C, G or T, R is A or G,

or comprising the following sequence SEQ ID N^(o)5 (positions 6247 to6269 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GGN YAY ATR GGN ATR GTN TGA GC

in which N is A, C, G or T, Y is C or T, R is A or G,

or constituted by the following sequence SEQ ID N^(o)6 (positions 6253to 6269 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): RGG NAT RGT NTG AGC

in which R is A or G, N is A, C, G or T, or those

(3)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)7 (positions 6556 to6580 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAY GTW GTN GCN CAY TTY CAC TAC G

in which Y is C or T, W is A or T, N is A, C, G or T,

or comprising the following sequence SEQ ID N^(o)8 (positions 6556 to6575 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAY GTW GTN GCN CAY TTY CA

in which Y is C or T, W is A or T, N is A, C, G or T,

or constituted by the following sequence SEQ ID N^(o)9 (positions 6556to 6570 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TAY GTW GTN GCN CAY

in which Y is C or T, W is A or T, N is A, C, G or T,

or those

(4)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)10 (positions 6131 to6154 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TRT AYC ARC AYY TRT TYT GRT TCT K

in which R is A or G, Y is C or T, K is G or T,

or comprising the following sequence SEQ ID N^(o)11 (positions 6134 to6154 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): ACC AAC ACT TAT TCT GAT TCT

or constituted by the following sequence SEQ ID N^(o)12 (positions 6139to 6154 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): AYY AAC ACT TAT TCT

in which Y is C or T,

or those

(5)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)13 (positions 6277 to6303 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): CYA TYG GMC TYT YGG YTT TAT YGT V

in which Y is C or T, M is A or C, V is A, C or G,

or comprising the following sequence SEQ ID N^(o)14 (positions 6283 to6303 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GGC CTC CTT GGC TTT ATT GTA

or constituted by the following sequence SEQ ID N^(o)15 (positions 6288to 6303 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): YTY GGY TTT ATT GTV

in which Y is C or T, V is A, C or G,

or those

(6)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)16 (positions 6496 to6522 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GGM YTW ACA GGN ATY RTH YTR GCY AA

in which M is A or C, W is A or T, Y is C or T, N is A, C, G or T, R isA or G, H is A, C or T,

or comprising the following sequence SEQ ID N^(o)17 (positions 6496 to6519 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GGC TTA ACA GGA ATT GTA CTA GCT

or constituted by the following sequence SEQ ID N^(o)18 (positions 6496to 6510 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GGC TTA ACA GGA ATT

or those

(7)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)19 (positions 6195 to6219 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): CGG RAT AAT YTC YCA YAT YGT AGC C

in which R is A or G, Y is C or T,

or comprising the following sequence SEQ ID N^(o)20 (positions 6200 to6219 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAA TYT CYC AYA TYG TAG CC

in which Y is C or T,

or constituted by the following sequence SEQ ID N^(o)21 (positions 6205to 6219 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TCY CAY ATY GTA GCC

in which Y is C or T,

or those

(8)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)22 (positions 6324 to6348 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): AGT BGG RAT RGA YGT DGA YAC MCG T

in which B is C, G or T, R is A or G, Y is C or T, D is A, G or T, M isA or C,

or comprising the following sequence SEQ ID N^(o)23 (positions 6329 to6348 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GRA TRG AYG TDG AYA CMC GT

in which R is A or G, Y is C or T, D is A, G or T, M is A or C,

or constituted by the following sequence SEQ ID N^(o)24 (positions 6334to 6348 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GAY GTD GAY ACM CGT

in which Y is C or T, D is A, G or T, M is A or C,

or those

(9)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)25 (positions 6498 to6523 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): ACT TAC AGG NAT YRT HCT RGC YAA YT

in which N is A, C, G or T, Y is C or T, R is A or G, H is A, C or T,

or comprising the following sequence SEQ ID N^(o)26 (positions 6498 to6517 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): ACT TAG AGG NAT YRT HCT RG

in which N is A, C, G or T, Y is C or T, R is A or G, H is A, C or T,

or constituted by the following sequence SEQ ID N^(o)27 (positions 6498to 6512 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): ACT TAC AGG NAT YRT

in which N is A, C, G or T, Y is C or T, R is A or G,

or those

(10)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)28 (positions 6399 to6423 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): AGT YTT YAG YTG AYT AGC AAC YYT V

in which Y is C or T, V is A, C or G,

or comprising the following sequence SEQ ID N^(o)29 (positions 6404 to6423 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TTA GCT GAT TAG CAA CTT TA

or constituted by the following sequence SEQ ID N^(o)30 (positions 6409to 6423 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TGA YTA GCA ACY YTV

in which Y is C or T, V is A, C or G,

or those

(11)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)31 (positions 6552 to6577 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): RTA YTA YGT ACT MGC YCA YTT YCA CT

in which R is A or G, Y is C or T, M is A or C,

or comprising the following sequence SEQ ID N^(o)32 (positions 6552 to6572 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GTA TTA CGT AGT AGC CCA TT

or constituted by the following sequence SEQ ID N^(o)33 (positions 6552to 6566 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): RTA YTA YGT ACT MGC

in which R is A or G, Y is C or T, M is A or C,

or those

(12)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)34 (positions 6237 to6261 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): AGA RCC NTT YGG RYA YAT RGG HAT R

in which R is A or G, N is A, C, G or T, Y is C or T, H is A, C or T,

or comprising the following sequence SEQ ID N^(o)35 (positions 6242 to6261 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): CCT TTG GAT ATA TAG GCA TG

or constituted by the following sequence SEQ ID N^(o)36 (positions 6248to 6261 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GGR YAY ATR GGH ATR

in which R is A or G, Y is C or T, H is A, C or T,

or those

(13)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)37 (positions 6381 to6406 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): YAT YCC RAC AGG YGT WAA AGT YTT YA

in which Y is C or T, R is A or G, W is A or T,

or comprising the following sequence SEQ ID N^(o)38 (positions 6381 to6400 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAT CCC AAC AGG TGT AAA AG

or constituted by the following sequence SEQ ID N^(o)39 (positions 6381to 6395 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TAT YCC RAG AGG YGT

in which Y is C or T, R is A or G,

or those

(14)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)40 (positions 6267 to6291 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): AGC YAT RAT RGC YAT YGG MCT YCT Y

in which Y is C or T, R is A or G, M is A or C,

or comprising the following sequence SEQ ID N^(o)41 (positions 6272 to6291 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TGA TGG CTA TTG GCC TCC TC

or constituted by the following sequence SEQ ID N^(o)42 (positions 6277to 6291 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GCY ATY GGM CTY CTY

in which Y is C or T, M is A or C,

or those

(15)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)43 (positions 6451 to6475 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): HCC BMT MCT BTG RGC CCT V GG YTT YA

in which H is A, C or T, B is C, G or T, M is A or C, R is A or G, V isA, C or G, Y is C or T,

or comprising the following sequence SEQ ID N^(o)44 (positions 6451 to6469 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): CCC TCT ACT CTG AGC CCT AG

or constituted by the following sequence SEQ ID N^(o)45 (positions 6451to 6464 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): HCC BMT MCT BTG RGC

in which H is A, C or T, B is C, G or T, M is A or C, R is A or G,

or those

(16)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)46 (positions 6194 to6219 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TSG RAT AAT YTC YCA YAT YGT AGC V

in which S is C or G, R is A or G, Y is C or T, V is A, C or G,

or comprising the following sequence SEQ ID N^(o)47 (positions 6200 to6219 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAA TTT CTC ACA TCG TAG CG

or constituted by the following sequence SEQ ID N^(o)48 (positions 6205to 6219 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TCY CAY ATY GTA GCV

in which Y is C or T, V is A, C or G,

or those

(17)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)49 (positions 6342 to6366 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): YAC MCG WGC HTA CTT YAC ATC YGC A

in which Y is C or T, M is A or C, W is A or T, H is A, C or T

or comprising the following sequence SEQ ID N^(o)50 (positions 6342 to6361 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAC ACG TGC CTA CTT TAC AT

or constituted by the following sequence SEQ ID N^(o)51 (positions 6342to 6356 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): YAC MCG WGC HTA CTT

in which Y is C or T, M is A or C, W is A or T, H is A, C or T,

or those

(18)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)52 (positions 6152 to6177 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TCT KCG GNC AYC CYG AAG THT AYA TH

in which K is G or T, N is A, C, G or T, Y is C or T, H is A, C or T,

or comprising the following sequence SEQ ID N^(o)53 (positions 6158 to6177 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GAC ACC CCG AAG TAT ACA TA

or constituted by the following sequence SEQ ID N^(o)54 (positions 6163to 6177 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): CCY GAA GTH TAY ATH

in which Y is C or T, H is A, C or T,

or those

(19)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)55 (positions 6303 to6328 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): VTG RGC YCA YCA CAT RTT YAC AGT BG

in which V is A, C or G, R is A or G, Y is C or T, B is C, G or T,

or comprising the following sequence SEQ ID N^(o)56 (positions 6303 to6322 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GTG AGC CCA TCA CAT GTT TA

or constituted by the following sequence SEQ ID N^(o)57 (positions 6303to 6317 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): VTG RGC YCA YCA CAT,

in which V is A, C or G, R is A or G, Y is C or T.

The oligonucleotides or primers as defined above allow the detection andoptionally the identification of the DNA fragments originating fromgadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae.

A subject of the invention is also the pairs of primers constituted:

by any one of the oligonucleotides SEQ ID N^(o)1, SEQ ID N^(o)2, SEQ IDN^(o)3, SEQ ID N^(o)4, SEQ ID N^(o)5, SEQ ID N^(o)6, and any one of theoligonucleotides SEQ ID N^(o)7, SEQ ID N^(o)8, SEQ ID N^(o)9 as definedabove or,

by any one of the oligonucleotides SEQ ID N^(o)10, SEQ ID N^(o)11, SEQID N^(o)12, SEQ ID N^(o)13, SEQ ID N^(o)14, SEQ ID N^(o)15, and any oneof the oligonucleotides SEQ ID N^(o)16, SEQ ID N^(o)17, SEQ ID N^(o)18as defined above or,

by any one of the oligonucleotides SEQ ID N^(o)19, SEQ ID N^(o)20, SEQID N^(o)21, SEQ ID N^(o)22, SEQ ID N^(o)23, SEQ ID N^(o)24, and any oneof the oligonucleotides SEQ ID N^(o)25, SEQ ID N^(o)26, SEQ ID N^(o)27as defined above or,

by any one of the oligonucleotides SEQ ID N^(o)28, SEQ ID N^(o)29, SEQID N^(o)30, and any one of the oligonucleotides SEQ ID N^(o)31, SEQ IDN^(o)32, SEQ ID N^(o)33 as defined above or,

by any one of the oligonucleotides SEQ ID N^(o)34, SEQ ID N^(o)35, SEQID N^(o)36, and any one of the oligonucleotides SEQ ID N^(o)37, SEQ IDN^(o)38, SEQ ID N^(o)39 as defined above or,

by any one of the oligonucleotides SEQ ID N^(o)40, SEQ ID N^(o)41, SEQID N^(o)42, and any one of the oligonucleotides SEQ ID N^(o)43, SEQ IDN^(o)44, SEQ ID N^(o)45 as defined above or,

by any one of the oligonucleotides SEQ ID N^(o)46, SEQ ID N^(o)47, SEQID N^(o)48, and any one of the oligonucleotides SEQ ID N^(o)49, SEQ IDN^(o)50, SEQ ID N^(o)51 as defined above or,

by any one of the oligonucleotides SEQ ID N^(o)52, SEQ ID N^(o)53, SEQID N^(o)54, and any one of the oligonucleotides SEQ ID N^(o)55, SEQ IDN^(o)56, SEQ ID N^(o)57 as defined above,

and advantageously constituted by the pair of oligonucleotides chosenfrom the following pairs:

(SEQ ID N^(o)2 and SEQ ID N^(o)8),

(SEQ ID N^(o)5 and SEQ ID N^(o)8),

(SEQ ID N^(o)11 and SEQ ID N^(o)17),

(SEQ ID N^(o)14 and SEQ ID N^(o)17),

(SEQ ID N^(o)20 and SEQ ID N^(o)26),

(SEQ ID N^(o)23 and SEQ ID N^(o)26),

(SEQ ID N^(o)29 and SEQ ID N^(o)32),

(SEQ ID N^(o)35 and SEQ ID N^(o)38),

(SEQ ID N^(o)41 and SEQ ID N^(o)44),

(SEQ ID N^(o)47 and SEQ ID N^(o)50),

(SEQ ID N^(o)53 and SEQ ID N^(o)56).

The pairs of primers (SEQ ID N^(o)2 and SEQ ID N^(o)8) and (SEQ IDN^(o)5 and SEQ ID N^(o)8) as defined above each allow the obtaining, byPCR reaction, of at least one DNA fragment originating from fishbelonging to the order of the gadiformes, displaying a determined sizeand sequence, specific to the gadiformes.

The pairs of primers (SEQ ID N^(o)11 and SEQ ID N^(o)17) and (SEQ IDN^(o)14 and SEQ ID N^(o)17) as defined above each allow the obtaining,by PCR reaction, of at least one DNA fragment originating from fishbelonging to the order of the gadiformes, and in particular to thefamily of the gadidae, displaying a determined size and sequence,specific to the gadidae.

The pairs of primers (SEQ ID N^(o)20 and SEQ ID N^(o)26) and (SEQ IDN^(o)23 and SEQ ID N^(o)26) as defined above each allow the obtaining,by PCR reaction, of at least one DNA fragment originating from fishbelonging to the order of the gadiformes, and in particular to thefamily of the merluccidae, displaying a determined size and sequence,specific to the merluccidae.

The pair of primers (SEQ ID N^(o)29 and SEQ ID N^(o)32) as defined aboveallows the obtaining, by PCR reaction, of at least one DNA fragmentoriginating from fish belonging to the order of the gadiformes, and inparticular to the family of the gadidae, displaying a determined sizeand sequence, specific to the species Gadus morhua (Atlantic cod).

The pair of primers (SEQ ID N^(o)35 and SEQ ID N^(o)38) as definedabove, allows the obtaining, by PCR reaction, of at least one DNAfragment originating from fish belonging to the order of the gadiformes,and in particular to the family of the gadidae, displaying a determinedsize and sequence, specific to the species Pollachius virens (pollock).

The pair of primers (SEQ ID N^(o)41 and SEQ ID N^(o)44) as defined aboveallows the obtaining, by PCR reaction, of at least one DNA fragmentoriginating from fish belonging to the order of the gadiformes, and inparticular to the family of the gadidae, displaying a determined sizeand sequence, specific to the species Theragra chalcogramma (Alaskanpollock).

The pair of primers (SEQ ID N^(o)47 and SEQ ID N^(o)50) as defined aboveallows the obtaining, by PCR reaction, of at least one DNA fragmentoriginating from fish belonging to the order of the gadiformes, and inparticular to the family of the gadidae, displaying a determined sizeand sequence, specific to the species Melanogrammus aeglefinus(haddock).

The pair of primers (SEQ ID N^(o)53 and SEQ ID N^(o)56) as defined aboveallows the obtaining, by PCR reaction, of at least one DNA fragmentoriginating from fish belonging to the order of the gadiformes, and inparticular to the family of the gadidae, displaying a determined sizeand sequence, specific to the species Merlangius merlangus (whiting).

A subject of the invention is also the DNA fragments as amplified at theend of the method as described above, comprising approximately 100 toapproximately 500 base pairs.

A DNA fragment of the invention advantageously displays a sequenceidentity of at least 80%, preferably 90% and advantageously 95% with atleast one of the sequences contained in:

the following SEQ ID N^(o)58: AYCARCAYYT RTTCTGATTC TKCGGNCAYCCYGAAGTHTA YATHCTNATY YTMCCHGGMT TCGGRATAAT YTCYCAYATY GTAGCVTAYTAYTCAGGNAA RMAAGARCCN TTYGGRYAYA TRGGHATRGT NTGAGCYATR ATRGCYATYGGMCTYCTYGG YTTTATYGTV TGRGCYCAYC ACATRTTYAC AGTBGGRATR GAYGTDGAYACMCGWGCHTA CTTYACATCY GCAACBATAA TYATYGCYAT YCCRACAGGY GTWAAAGTYTTYAGYTGAYT AGCAACYYTV CAYGGRGCCT CARTTAARTG RGAVACHCCB MTMCTBTGRGCCCTDGGYTT YATYTTYCTM TTYACMGTHG GVGGMYTWAC AGGNATYRTH YTRGCYAAYTCYTCYCTAGA YATYGTDCTY CAYGAYACRT AYTAMGTAGT MGCYCAYTTY CA

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T,

said sequence SEQ ID N^(o) 58 comprising 442 base pairs,

or the following SEQ ID N^(o)59: GGRYAYATRG GHATRGTNTG AGCYATRATRGCYATYGGMC TYCTYGGYTT TATYGTVTGR GCYCAYCACA TRTTYACAGT BGGRATRGAYGTDGAYACMC GWGCHTACTT YACATCYGCA ACBATAATYA TYGCYATYCC RACAGGYGTWAAAGTYTTYA GYTGAYTAGC ACYYTVCAYG GRGGCTCART TAARTGRGAV ACHCCBMTMCTBTGRGCCCT DGGYTTYATY TTYCTMTTYA CMGTHGGVGG MYTWACAGGN ATYRTHYTRGCYAAYTCYTC YCTAGAYATY GTDCTYCAYG AYACRTAYTA MGTAGTMGCY CAYTTYCAin which R is A or G, Y is C or T, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T,

said sequence SEQ ID N^(o) 59 comprising 328 base pairs,

or the following SEQ ID N^(o)60: AYCARCAYYT RTTCTGATTC TKCGGNCAYCCYGAAGTHTA YATHCTNATY YTMCCHGGMT TCGGRATAAT YTCYCAYATY GTAGCVTAYTAYTCAGGNAA RMAAGARCCN TTYGGRYAYA TRGGHATRGT NTGAGCYATR ATRGCYATYGGMCTYCTYGG YTTTATYGTV TGRGCYCAYC ACATRTTYAC AGTBGGRATR GAYGTDGAYACMCGWGCHTA CTTYACATCY GCAACBATAA TYATYGCYAT YCCRACAGGY GTWAAAGTYTTYAGYTGAYT AGCAACYYTV CAYGGRGGCT CARTTAARTG RGAVACHCCB MTMCTBTGRGCCCTDGGYTT YATYTTYCTM TTYACMGTHG GVGGMYTWAC AGGNATYRTH YTRGCY

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T,

said sequence SEQ ID N^(o)60 comprising 386 base pairs,

or the following SEQ ID N^(o)61: GGMCTYCTYG GYTTTATYGT VTGRGCYCAYCACATRTTYA CAGTBGGRAT RGAYGTDGAY ACMCGWGCHT ACTTYACATC YGCAACBATAATYATYGCYA TYCCRACAGG YGTWAAAGTY TTYAGYTGAY TAGCAACYYT VCAYGGRGGCTCARTTAART GRGAVACHCC BMTMCTBTGR GCCCTDGGYT TYATYTTYCT MTTYACMGTHGGVGGMYTWA CAGGNATYRT HYTRGCY

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T,

said sequence SEQ ID N^(o) 61 comprising 237 base pairs,

or the following SEQ ID N^(o)62: TAATYTCYCA YATYGTAGCV TAYTAYTCAGGNAARMAAGA RCCNTTYGGR YAYATRGGHA TRGTNTGAGC YATRATRGCY ATYGGMCTYCTYGGYTTTAT YGTVTGRGCY CAYCACATRT TYACAGTBGG RATRGAYGTD GAYACMCGWGCHTACTTYAC ATCYGCAACB ATAATYATYG CYATYCCRAC AGGYGTWAAA GTYTTYAGYTGAYTAGCAAC YYTVCAYGGR GGCTCARTTA ARTGRGAVAC HCCBMTMCTB TGRGCCCTDGGYTTYATYTT YCTMTTYACM GTHGGVGGMY TWACAGGNAT YRTHYTRGin which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T,

said sequence SEQ ID N^(o) 62 comprising 318 base pairs,

or the following SEQ ID N^(o)63: GRATRGAYGT DGAYACMCGW GCHTACTTYACATCYGCAAC BATAATYATY GCYATYCCRA CAGGYGTWAA AGTYTTYAGY TGAYTAGCAACYYTVCAYGG RGGCTCARTT AARTGRGAVA CHCCBMTMCT BTGRGCCCTD GGYTTYATYTTYCTMTTYAC MGTHGGVGGM YTWACAGGNA TYRTHYTRG

in which R is A or G, Y is C or T, D is A, G or T, M is A or C, W is Aor T, B is C, G or T, V is A, C or G, H is A, C or T, N is A, C, G or T,

said sequence SEQ ID N^(o)63 comprising 189 base pairs,

or the following SEQ ID N^(o)64: TYAGYTGAYT AGCAACYYTV CAYGGRGGCTCARTTAARTG RGAVACHCCB MTMCTBTGRG CCCTDGGYTT YATYTTYCTM TTYACMGTHGGVGGMYTWAC AGGNATYRTH YTRGCYAAYT CYTCYCTAGA YATYGTDCTY CAYGAYACRTAYTAMGTAGT MGCYCAYT

in which Y is C or T, V is A, C or G, R is A or G, B is C, G or T, H isA, C or T, M is A or C, D is A, G or T, N is A, C, G or T,

said sequence SEQ ID N^(o)64 comprising 168 base pairs,

or the following SEQ ID N^(o)65: CNTTYGGRYA YATRGGHATR GTNTGAGCYATRATRGCYAT YGGMCTYCTY GGYTTTATYG TVTGRGCYCA YCACATRTTY ACAGTBGGRATRGAYGTDGA YACMCGWGCH TACTTYACAT CYGCAACBAT AATYATYGCY ATYCCRACAGGYGTWAAAG

in which N is A, C, G or T, R is A or G, Y is C or T, H is A, C or T, Mis A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T,

said sequence comprising 159 base pairs,

or the following sequence SEQ ID N^(o)66: TRATRGCYAT YGGMCTYCTYGGYTTTATYG TVTGRGCYCA YCACATRTTY ACAGTBGGRA TRGAYGTDGA YACMCGWGCHTACTTYACAT CYGCAACBAT AATYATYGCY ATYCCRACAG GYGTWAAAGT YTTYAGYTGAYTAGCAACYY TVCAYGGRGG CTCARTTAAR TGRGAVACHC CBMTMCTBTG RGCCCTDG

in which R is A or G, Y is C or T, M is A or C, V is A, C or G, B is C,G or T, D is A, G or T, W is A or T, H is A, C or T,

said SEQ ID N^(o)66 comprising 198 base pairs,

the following SEQ ID N^(o)67: TAATYTCYCA YATYGTAGCV TAYTAYTCAGGNAARMAAGA RCCNTTYGGR YAYATRGGHA TRGTNTGAGC YATRATRGCY ATYGGMCTYCTYGGYTTTAT YGTVTGRGCY CAYCACATRT TYACACTBGG RATRGAYGTD GAYACMCGWGCHTACTTYAC AT

in which Y is C or T, V is A, C or G, N is A, C, G or T, R is A or G, Mis A or C, H is A, C or T, B is C, G or T, D is A, G or T, W is A or T,

said sequence SEQ ID N^(o)67 comprising 162 base pairs,

or the following SEQ ID N^(o)68: GNCAYCCYGA AGTHTAYATH CTNATYYTMCCHGGMTTCGG RATAATYTCY CAYATYGTAG CVTAYTAYTC AGGNAARMAA GARCCNTTYGGRYAYATRGG HATRGTNTGA GCYATRATRG CYATYGGMCT YCTYGGYTTT ATYGTVTGRGCYCAYCACAT RTTYA

in which N is A, C, G or T, Y is C or T, H is A, C or T, M is A or C, Ris A or G, V is A, C or G,

said sequence SEQ ID N^(o) 68 comprising 165 base pairs.

The DNA fragments SEQ ID N^(o)58 and SEQ ID N^(o)59 as defined above arespecific to fish belonging to the order of the gadiformes. The DNAfragments SEQ ID N^(o)60 and SEQ ID N^(o)61 as defined above arespecific to fish belonging to the order of the gadiformes, and moreparticularly to fish belonging to the family of the gadidae. The DNAfragments SEQ ID N^(o)62 and SEQ ID N^(o)63 as defined above arespecific to fish belonging to the order of the gadiformes, and moreparticularly to fish belonging to the family of the merluccidae.

The DNA fragment SEQ ID N^(o)64 as defined above is specific to fishbelonging to the order of the gadiformes, more particularly to fishbelonging to the family of the gadidae, and more particularly thespecies Gadus morhua (Atlantic cod). The DNA fragment SEQ ID N^(o)65 asdefined above is specific to fish belonging to the order of thegadiformes, more particularly to fish belonging to the family of thegadidae, and more particularly to the species Pollachius virens(pollock). The DNA fragment SEQ ID N^(o)66 as defined above is specificto fish belonging to the order of the gadiformes, more particularly tofish belonging to the family of the gadidae, and more particularly tothe species Theragra chalcogramma (Alaskan pollock). The DNA fragmentSEQ ID N^(o)67 as defined above is specific to fish belonging to theorder of the gadiformes, more particularly to fish belonging to thefamily of the gadidae, and more particularly to the speciesMelanogrammus aeglefinus (haddock). The DNA fragment SEQ ID N^(o)68 asdefined above is specific to fish belonging to the order of thegadiformes, more particularly to fish belonging to the family of thegadidae, and more particularly to the species Merlangius merlangus(whiting).

According to an advantageous embodiment of the method of the invention,the obtained amplified DNA fragment(s) contained in the amplificationproduct is (are) identified:

by sequencing of at least one amplified DNA fragment contained in theamplification product, and in particular of one amplified DNA fragmentor,

directly, by visualization of the presence of the amplification productby gel electrophoresis.

According to an advantageous embodiment of the invention, the method ofidentification by sequencing of at least one obtained amplified DNAfragment allows the identification of the fish belonging to the order ofthe gadiformes, in particular chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae.

The method of direct identification by simple visualization of thepresence of the amplification product allows the identification of thespecies of fish belonging to the order of the gadiformes, and moreparticularly of the species of fish belonging to the family of thegadidae.

Each of the two methods of identification is described in more detailbelow.

1) The method of identification by sequencing of at least one obtainedamplified DNA fragment is called <<global strategy>> in the following,as it allows the detection and identification of the presence or theabsence of all the existing gadiformes. By way of guidance, a list ofgadiformes that are able to be detected and identified by the method ofthe invention is given in the table 1 below. This list is notexhaustive, however.

The primers used in the global strategy in order to effect theamplification by the PCR method of at least one DNA sequence or fragmentspecific to the genome of the gadiformes, possibly present in a sampleof organic material to be analysed, are the 27 primers listed below andas defined above:

SEQ ID N^(o)1, SEQ ID N^(o)2, SEQ ID N^(o)3, SEQ ID N^(o)4, SEQ IDN^(o)5, SEQ ID N^(o)6, SEQ ID N^(o)7, SEQ ID N^(o)8, SEQ ID N^(o)9, SEQID N^(o)10, SEQ ID N^(o)11, SEQ ID N^(o)12, SEQ ID N^(o)13, SEQ IDN^(o)14, SEQ ID N^(o)15, SEQ ID N^(o)16, SEQ ID N^(o)17, SEQ ID N^(o)18,SEQ ID N^(o)19, SEQ ID N^(o)20, SEQ ID N^(o)21, SEQ ID N^(o)22, SEQ IDN^(o)23, SEQ ID N^(o)24, SEQ ID N^(o)25, SEQ ID N^(o)26 and SEQ IDN^(o)27,

and more particularly the 9 primers SEQ ID N^(o)2, SEQ ID N^(o)5, SEQ IDN^(o)8, SEQ ID N^(o)11, SEQ ID N^(o)14, SEQ ID N^(o)17, SEQ ID N^(o)20,SEQ ID N^(o)23 and SEQ ID N^(o)26.

The pairs of primers advantageously used are as defined above.

According to a particularly advantageous implementation of the presentinvention, a part of the stages of hybridization of the cyclesconstituting the amplification reaction is carried out at a temperatureof approximately 50° C. to approximately 58° C. Moreover, the applicantfound that a temperature of 55° C. was particularly suitable forobtaining a specific amplification. Such a mode of implementation allowsa greater specificity of amplification to be achieved.

It will be noted on this point that the applicant has solved a certainnumber of technical problems for the implementation of this method.First of all, the choice of primers constituted a real problem, as itwas necessary to select on the one hand sequences common to thedifferent species of gadiformes but not to other species of fish, and onthe other hand hybridizing in stable manner, in very variablephysico-chemical conditions, representative of the great variability ofthe organic materials likely to contain biological materials originatingfrom gadiformes. The temperatures of the stages of separation of thestrands and of elongation are advantageously respectively approximately94° C. and approximately 72° C.

The method described above is specific to the gadiformes but globalbetween all the species of gadiformes as it does not give anamplification reaction detectable in the presence of DNA of an originother than the gadiformes.

According to an advantageous embodiment of the method of the invention(global strategy), the use of the following pairs of oligonucleotideprimers:

(SEQ ID N^(o)2 and SEQ ID N^(o)8),

(SEQ ID N^(o)5 and SEQ ID N^(o)8),

(SEQ ID N^(o)11 and SEQ ID N^(o)17),

(SEQ ID N^(o)14 and SEQ ID N^(o)17),

(SEQ ID N^(o)20 and SEQ ID N^(o)26) and

(SEQ ID N^(o)23 and SEQ ID N^(o)26),

allows respectively the obtaining of:

a DNA fragment SEQ ID N^(o)58 as defined above,

a DNA fragment SEQ ID N^(o)59 as defined above,

a DNA fragment SEQ ID N^(o)60 as defined above,

a DNA fragment SEQ ID N^(o)61 as defined above,

a DNA fragment SEQ ID N^(o)62 as defined above and,

a DNA fragment SEQ ID N^(o)63 as defined above.

The DNA fragments described above, obtained at the end of the PCRreaction, can be detected even when a substantial fraction of the DNA isdegraded, namely after action by physical, chemical and/or biochemicalfactors, and during various transformations of the samples of organicmaterial to be analysed.

The described method displays a great simplicity of interpretation,because of the production of a single and unique amplification product,specific to the DNA of gadiformes and which therefore is not found inthe DNA amplifications products of other orders. The uniqueness of theamplification product obtained at the end of the PCR reaction representsanother advantage of the present invention, that of allowing of asubstantial sensitivity to be achieved and greatly facilitating theinterpretation of the results. The method according to the presentinvention offers a large number of advantages compared with alreadyknown identification techniques.

The amplification product can be shown by any method known to a personskilled in the art, and in particular by simple agarose gelelectrophoresis. The reading of the migration profiles of theamplification product obtained with the method of the inventiontherefore simply consists of determining the presence of a single andunique migration band in an electrophoresis gel. In the absence of sucha band, it can be considered that there are no detectable traces of DNAof gadiformes. The presence of a band signifies on the other hand thatthe DNA of gadiformes is present in the sample, and therefore that thesample in question contains biological material based on gadiformes.

When the obtained amplification product comprises several copies of thesame amplified DNA fragment or sequence, this latter can then besequenced in order to determine its nucleotide sequence. The comparisonof the nucleotide sequence obtained with all the known nucleotidesequences of the gadiformes allows the determination of the species ofgadiforme present in the sample of organic material, and thus thedifferentiation of the species from one another.

When the obtained amplification product comprises several copies ofdifferent amplified DNA fragments or sequences, the sequencing of eachof the different amplified DNA fragments will be preceded by a cloningmethod. Thus, according to an advantageous embodiment of the method ofthe invention, the sequencing of each of the different amplified DNAfragments is preceded by a cloning method when the sample of organicmaterial comprises a mixture of different DNA fragments originating fromdifferent species of gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae, saidcloning method permitting the separation from said mixture of thedifferent DNA fragments originating from the different species of fish.

According to an advantageous embodiment of the method of the invention,the presence of DNA originating from gadiformes in a sample of organicmaterial is detected by amplification of at least one DNA sequencespecific to the genome of the gadiformes using any one of theoligonucleotides SEQ ID N^(o)1 SEQ ID N^(o)2, SEQ ID N^(o)3, SEQ IDN^(o)4, SEQ ID N^(o)5, SEQ ID N^(o)6, and any one of theoligonucleotides SEQ ID N^(o)7, SEQ ID N^(o)8, SEQ ID N^(o)9 as definedabove,

and advantageously using the pair of oligonucleotides (SEQ ID N^(o)2 andSEQ ID N^(o)8) or the pair of oligonucleotides (SEQ ID N^(o)5 and SEQ IDN^(o)8), in order to obtain respectively at least one of the DNAsequences contained in SEQ ID N^(o)58 or in SEQ ID N^(o)59 as definedabove, said sequences being specific to the genome of the gadiformes,

and in that at least one species of gadiforme present in said sample oforganic material is identified by sequencing of at least one of the DNAsequences contained in SEQ ID N^(o)58 or in SEQ ID N^(o)59, saidsequencing being optionally preceded by a cloning method when theamplification product obtained at the end of the PCR reaction comprisesa mixture of different DNA fragments or sequences originating fromdifferent species of gadiformes.

Thus, the pairs of oligonucleotides (SEQ ID N^(o)2 and SEQ ID N^(o)8) or(SEQ ID N^(o)5 and SEQ ID N^(o)8) allow, by amplification followed by asequencing, the detection and identification of all the existinggadiformes, whatever the family concerned (gadidae, merluccidae,macrouridae etc.).

According to another advantageous embodiment of the method of theinvention, the presence of DNA originating from gadidae in a sample oforganic material is detected by amplification of at least one DNAsequence specific to the genome of the gadidae using any one of theoligonucleotides SEQ ID N^(o)10, SEQ ID N^(o)11, SEQ ID N^(o)12, SEQ IDN^(o)13, SEQ ID N^(o)14, SEQ ID N^(o)15, and any one of theoligonucleotides SEQ ID N^(o)16, SEQ ID N^(o)17, SEQ ID N^(o)18 asdefined above,

and advantageously using the pair of oligonucleotides (SEQ ID N^(o)11and SEQ ID N^(o)17) or the pair of oligonucleotides (SEQ ID N^(o)14 andSEQ ID N^(o)17), in order to obtain respectively at least one of the DNAsequences contained in SEQ ID N^(o)60 or in SEQ ID N^(o)61 as definedabove, said sequences being specific to the genome of the gadidae,

and in that at least one species of gadidae present in said sample oforganic material is identified by sequencing of at least one of the DNAsequences contained in SEQ ID N^(o)60 or in SEQ ID N^(o)61, saidsequencing being optionally preceded by a cloning method when theamplification product obtained at the end of the PCR reaction comprisesa mixture of different DNA fragments or sequences originating fromdifferent species of gadidae.

Thus the pairs of oligonucleotides (SEQ ID N^(o)11 and SEQ ID N^(o)17)or (SEQ ID N^(o)14 and SEQ ID N^(o)17) allow, by amplification followedby a sequencing, the detection and identification of all the existinggadidae, whatever the species or genus considered, but do not allow thedetection and identification of the gadiformes not belonging to thefamily of the gadidae.

According to another advantageous embodiment of the method of theinvention, the presence of DNA originating from merluccidae in a sampleof organic material is detected by amplification of at least one DNAsequence specific to the genome of the merluccidae using any one of theoligonucleotides SEQ ID N^(o)19, SEQ ID N^(o)20, SEQ ID N^(o)21, SEQ IDN^(o)22, SEQ ID N^(o)23, SEQ ID N^(o)24, and any one of theoligonucleotides SEQ ID N^(o)25, SEQ ID N^(o)26, SEQ ID N^(o)27 asdefined above,

and advantageously using the pair of oligonucleotides (SEQ ID N^(o)20and SEQ ID N^(o)26) or the pair of oligonucleotides (SEQ ID N^(o)23 andSEQ ID N^(o)26), in order to obtain respectively at least one of the DNAsequences contained in SEQ ID N^(o)62 or in SEQ ID N^(o)63 as definedabove, said sequences being specific to the genome of the merluccidae,

and in that at least one species of merluccidae present in said sampleof organic material is identified by sequencing of at least one of theDNA sequences contained in SEQ ID N^(o)62 or in SEQ ID N^(o)63, saidsequencing being optionally preceded by a cloning method when theamplification product obtained at the end of the PCR reaction comprisesa mixture of different DNA fragments or sequences originating fromdifferent species of merluccidae.

Thus, the pairs of oligonucleotides (SEQ ID N^(o)20 and SEQ ID N^(o)26)and (SEQ ID N^(o)23 and SEQ ID N^(o)26) allow, by amplification followedby a sequencing, the detection and identification of all the existingmerluccidae, whatever the species or genus considered, but do not allowthe detection and identification of the gadiformes not belonging to thefamily of the merluccidae.

2) The method of direct identification by simple visualization of thepresence of the amplification product using a gel electrophoresis iscalled <<specific strategy >> in the following, as it allows thedetection and direct identification of very specific species of gadidae.In the <<specific strategy >>, the stage of detection and ofidentification is a simultaneous stage.

In fact, this method, unlike the method described above (“globalstrategy”), does not require an additional identification stage at theend of the detection stage (the detection of the presence of biologicalmaterials originating from gadiformes being possible by the obtaining ofan amplification product), and thus allows the detection andidentification directly after amplification of certain very particularspecies of fish, which in the present case are species which are veryfamiliar through being much used in food or other preparations. Thespecies of fish that can be simultaneously detected and identifiedaccording to the method of the present invention are more particularlythe following five species of gadidae:

Gadus morhua (common or Atlantic cod),

Pollachius virens (pollock),

Theragra chalcogramma (Alaskan pollock),

Melanogrammus aeglefinus (haddock) and

Merlangius merlangus (whiting).

The amplification of the DNA is carried out by the polymerase chainamplification method (PCR) as described previously.

The specific strategy, allowing the simultaneous detection andidentification of the presence of very specific species of gadidae in asample of organic material, is described in more detail below.

(a) Specific strategy allowing the identification of Gadus morhua(common or Atlantic cod).

The primers used in the specific strategy in order to effect theamplification by the PCR method of a DNA sequence originating fromgadidae, and more particularly Gadus morhua, possibly present in asample of organic material to be analysed, are the 6 primers listedbelow and as defined above:

SEQ ID N^(o)28, SEQ ID N^(o)29, SEQ ID N^(o)30, SEQ ID N^(o)31, SEQ IDN^(o)32, SEQ ID N^(o)33, and more particularly the 2 primers SEQ IDN^(o)29 and SEQ ID N^(o)32.

According to an advantageous embodiment of the method of the invention(“specific strategy”), the use of the pair of oligonucleotides (SEQ IDN^(o)29 and SEQ ID N^(o)32) allows an amplification product containing aDNA fragment SEQ ID N^(o)64 as defined above to be obtained. Thepresence of said amplification product is detected by simplevisualization by agarose gel electrophoresis, and thus allows thesimultaneous detection and identification of the presence of Gadusmorhua (common or Atlantic cod).

The expression “amplification product containing a DNA fragment” used inparagraph 2) above and hereafter must be understood to mean“amplification product containing several copies of the same DNAfragment or sequence”.

(b) Specific strategy allowing the identification of Pollachius virens(pollock).

The primers used in the specific strategy in order to effect theamplification by the PCR method of a DNA sequence originating fromgadidae, and more particularly in Pollachius virens, possibly present ina sample of organic material to be analysed, are the 6 primers listedbelow and as defined above:

SEQ ID N^(o)34, SEQ ID N^(o)35, SEQ ID N^(o)36, SEQ ID N^(o)37, SEQ IDN^(o)38, SEQ ID N^(o)39, and more particularly the 2 primers SEQ IDN^(o)35 and SEQ ID N^(o)38.

According to an advantageous embodiment of the method of the invention,the use of the pair of oligonucleotides (SEQ ID N^(o)35 and SEQ IDN^(o)38), allows an amplification product containing a DNA fragment SEQID N^(o)65 as defined above to be obtained. The presence of saidamplification product is detected by simple visualization by agarose gelelectrophoresis, and thus allows the simultaneous detection andidentification of the presence of Pollachius virens (pollock).

(c) Specific strategy allowing the identification of Theragrachalcogramma (Alaskan pollock).

The primers used in the specific strategy in order to effect theamplification by the PCR method of a DNA sequence originating fromgadidae, and more particularly in Theragra chalcogramma, possiblypresent in a sample of organic material to be analysed, are the 6primers listed below and as defined above:

SEQ ID N^(o)40, SEQ ID N^(o)41, SEQ ID N^(o)42, SEQ ID N^(o)43, SEQ IDN^(o)44, SEQ ID N^(o)45, and more particularly the 2 primers SEQ IDN^(o)41 and SEQ ID N^(o)44.

According to an advantageous embodiment of the method of the invention,the use of the pair of oligonucleotides (SEQ ID N^(o)41 and SEQ IDN^(o)44) allows an amplification product containing a DNA fragment SEQID N^(o)66 as defined above to be obtained. The presence of saidamplification product is detected by simple visualization by agarose gelelectrophoresis, and thus allows the simultaneous detection andidentification of the presence of Theragra chalcogramma (Alaskanpollock).

According to a particularly advantageous implementation of the presentinvention, in the specific strategy allowing the identification ofrespectively Gadus morhua (common or Atlantic cod), Pollachius virens(pollock) and Theragra chalcogramma (Alaskan pollock), a part of thestages of hybridization of the cycles constituting the amplificationreaction is carried out at a temperature of 60° C. which is particularlysuitable for achieving a specific amplification. Such an implementationallows a greater specificity of amplification to be achieved.

(d) Specific strategy allowing the identification of Melanogrammusaeglefinus (haddock).

The primers used in the specific strategy in order to effect theamplification by the PCR method of a DNA sequence originating fromgadidae, and more particularly in Melanogrammus aeglefinus, possiblypresent in a sample of organic material to be analysed, are the 6primers listed below and as defined above:

SEQ ID N^(o)46, SEQ ID N^(o)47, SEQ ID N^(o)48, SEQ ID N^(o)49, SEQ IDN^(o)50, SEQ ID N^(o)51, and more particularly the 2 primers SEQ IDN^(o)47 and SEQ ID N^(o)50.

According to an advantageous embodiment of the method of the invention,the use of the pair of oligonucleotides (SEQ ID N^(o)47 and SEQ IDN^(o)50) allows an amplification product containing a DNA fragment SEQID N^(o)67 as defined above to be obtained. The presence of saidamplification product is detected by simple visualization by agarose gelelectrophoresis, and thus allows the simultaneous detection andidentification of the presence of Melanogrammus aeglefinus (haddock).

(e) Specific strategy allowing the identification of Merlangiusmerlangus (whiting).

The primers used in the specific strategy in order to effect theamplification by the PCR method of a DNA sequence originating fromgadidae, and more particularly in Merlangius merlangus, possibly presentin a sample of organic material to be analysed, are the 6 primers listedbelow and as defined above:

SEQ ID N^(o)52, SEQ ID N^(o)53, SEQ ID N^(o)54, SEQ ID N^(o)55, SEQ IDN^(o)56, SEQ ID N^(o)57, and more particularly the 2 primers SEQ IDN^(o)53 and SEQ ID N^(o)56.

According to an advantageous embodiment of the method of the invention,the use of the pair of oligonucleotides (SEQ ID N^(o)53 and SEQ IDN^(o)56), allows respectively an amplification product containing a DNAfragment SEQ ID N^(o)68 as defined above to be obtained. The presence ofsaid amplification product is detected by simple visualization by gelagarose electrophoresis, and thus allows the simultaneous detection andidentification of the presence of Merlangius merlangus (whiting).

According to a particularly advantageous implementation of the presentinvention, in the specific strategy allowing the identification ofrespectively Melanogrammus aeglefinus (haddock) and Merlangius merlangus(whiting), a part of the stages of hybridization of the cyclesconstituting the amplification reaction is carried out at a temperatureof 65° C. which is particularly suitable for obtaining a specificamplification. Such an implementation allows a greater specificity ofamplification to be achieved.

According to an advantageous embodiment of the method of the invention,the presence of DNA originating from gadiformes in a sample of organicmaterial, in particular in gadidae chosen from the group constituted bythe species Gadus morhua, Pollachius virens, Theragra chalcogramma,Melanogrammus aeglefinus and Merlangius merlangus, is detected and eachof the aforementioned species is identified by amplification of at leastone DNA sequence specific to the genome of each of the aforementionedspecies of gadidae, with the help respectively:

of any one of the oligonucleotides SEQ ID N^(o)28, SEQ ID N^(o)29, SEQID N^(o)30, and any one of the oligonucleotides SEQ ID N^(o)31, SEQ IDN^(o)32, SEQ ID N^(o)33 as defined above or,

of any one of the oligonucleotides SEQ ID N^(o)34, SEQ ID N^(o)35, SEQID N^(o)36, and any one of the oligonucleotides SEQ ID N^(o)37, SEQ IDN^(o)38, SEQ ID N^(o)39 as defined above or,

of any one of the oligonucleotides SEQ ID N^(o)40, SEQ ID N^(o)41, SEQID N^(o)42, and any one of the oligonucleotides SEQ ID N^(o)43, SEQ IDN^(o)44, SEQ ID N^(o)45 as defined above or,

of any one of the oligonucleotides SEQ ID N^(o)46, SEQ ID N^(o)47, SEQID N^(o)48, and any one of the oligonucleotides SEQ ID N^(o)49, SEQ IDN^(o)50, SEQ ID N^(o)51 as defined above or,

of any one of the oligonucleotides SEQ ID N^(o)52, SEQ ID N^(o)53, SEQID N^(o)54, and any one of the oligonucleotides SEQ ID N^(o)55, SEQ IDN^(o)56, SEQ ID N^(o)57 as defined above,

and advantageously with the help respectively:

of the pair of oligonucleotides (SEQ ID N^(o)29 and SEQ ID N^(o)32) or,

of the pair of oligonucleotides (SEQ ID N^(o)35 and SEQ ID N^(o)38) or,

of the pair of oligonucleotides (SEQ ID N^(o)41 and SEQ ID N^(o)44) or,

of the pair of oligonucleotides (SEQ ID N^(o)47 and SEQ ID N^(o)50) or,

of the pair of oligonucleotides (SEQ ID N^(o)53 and SEQ ID N^(o)56), inorder to obtain respectively at least one of the DNA sequences containedin:

SEQ ID N^(o)64 specific to the genome of Gadus morhua (Atlantic cod),

SEQ ID N^(o)65 specific to the genome of Pollachius virens (pollock),

SEQ ID N^(o)66 specific to the genome of Theragra chalcogramma (Alaskanpollock),

SEQ ID N^(o)67 specific to the genome of Melanogrammus aeglefinus(haddock),

SEQ ID N^(o)68 specific to the genome of Merlangius merlangus (whiting),

said SEQ ID N^(o)64, SEQ ID N^(o)65, SEQ ID N^(o)66, SEQ ID N^(o)67 andSEQ ID N^(o)68 being as defined above.

Thus the pairs of oligonucleotides defined above allow the detection anddirect identification by amplification of a very particular species ofgadidae.

According to an advantageous embodiment of the method of the invention(global strategy), when the experimenter suspects the presence ofseveral different species of gadiformes in the sample of organicmaterial to be analysed, the cloning method will be carried out directlyat the end of the stage of amplification by PCR reaction of the DNAextracted from the sample. At the end of the cloning method thesequencing of each of the different DNA fragments or sequencescorresponding respectively to one and the same species of gadiformeswill be carried out.

According to another advantageous embodiment of the method of theinvention (global strategy), when the experimenter suspects the presenceof a single species of gadiforme in the sample of organic material to beanalysed, the sequencing will be carried out directly at the end of thestage of amplification by PCR reaction of the DNA extracted from thesample. However if numerous indeterminations appear on the profileobtained at the end of the sequencing, this means that the sample oforganic material to be analysed contains at least two different speciesof gadiformes. In this case, it will be necessary to proceed with thecloning in order to then carry out the sequencing of each of thedifferent DNA fragments or sequences corresponding respectively to oneand the same species of gadiformes.

According to another advantageous embodiment of the method of theinvention (specific strategy), when the experimenter suspects thatseveral different species of gadiformes are present in the sample oforganic material to be analysed, he will successively carry out severalamplifications by PCR reaction, using each of the appropriate pairs ofoligonucleotide primers, in order to detect and to identify in a singlestage the presence of each of the species of gadiformes which hesuspects are present in the sample of organic material to be analysed(such as Gadus morhua, and/or Pollachius virens, and/or Theragrachalcogramma, and/or Melanogrammus aeglefinus, and/or Merlangiusmerlangus).

According to another advantageous embodiment of the method of theinvention (specific strategy), when the experimenter suspects that asingle species of gadiforme is present in the sample of organic materialto be analysed, he will carry out a single amplification by PCRreaction, using the appropriate pair of oligonucleotide primers, inorder to detect and to identify in a single stage the presence of thespecies of gadiforme which he suspects is present in the sample oforganic material to be analysed (such as Gadus morhua or Pollachiusvirens or Theragra chalcogramma or Melanogrammus aeglefinus, orMerlangius merlangus).

A subject of the invention is also the use of nucleotide sequenceschosen from the oligonucleotide primers as defined above or followingsequences: CGG GAT CCT GTT CTG ATT CTT GAT TTC C or CGA CGG GAT CCC AACACC TGT TTC GAT CAT CGC GGC AAC, or DNA fragments as defined above, forthe implementation of a method for detecting the presence of biologicalmaterials originating from gadiformes chosen from the group constitutedby the gadidae, the merluccidae, the macrouridae and/or the moridae, andoptionally of identifying at least one species of gadiformes chosen fromthe group constituted by the gadidae, the merluccidae, the macrouridaeand/or the moridae present, in a sample of organic material likely tocontain such biological materials, and in particular in fresh or indeedprocessed products such as agri-foodstuffs products, in particularfillet, soup, terrine, pâtés, fat, flour, fish-based preparations etc.

DESCRIPTION OF THE FIGURES

FIG. 1 represents an agarose gel coloured with ethidium bromide. Amarker measuring 20 kilo base pairs (Kpb) (M) is deposited on the gel.The DNAs are extracted from different samples of organic materials suchas pollock fillet (well 1), salted cod (well 2), a cooked dish withAlaskan pollock (well 3), fish rillettes (well 4) and fish soup (well5), and are deposited on the gel.

FIGS. 2-a and 2-b represent an agarose gel coloured with ethidiumbromide. A marker measuring 100 bp (M) is deposited on the gel. Theoligonucleotides of the present invention were tested beforehand onreference DNAs (DNA of gadiformes).

In FIG. 2-a, the DNAS of gadiformes (wells 1, 2) were amplified usingthe primers SEQ ID N^(o)2 and SEQ ID N^(o)8. The amplification productobtained, containing at least one fragment SEQ ID N^(o)58, migratesforming a band of 442 base pairs, specific to the genome of thegadiformes. The DNAs of gadiformes (wells 3, 4) were amplified using theprimers SEQ ID N^(o)5 and SEQ ID N^(o)8. The amplification productobtained, containing at least one fragment SEQ ID N^(o)59, migratesforming a band of 328 base pairs, specific to the genome of thegadiformes. Well 5 is a negative amplification control.

In FIG. 2-b the DNAs of gadiformes (wells 2, 3) were amplified using theprimers SEQ ID N^(o)20 and SEQ ID N^(o)26. The amplification productobtained, containing at least one fragment SEQ ID N^(o)62, migratesforming a band of 318 base pairs, specific to the genome of themerluccidae. Well 1 is a negative amplification control. The DNAs ofgadiformes (wells 5, 6) were amplified using the primers SEQ ID N^(o)23and SEQ ID N^(o)26. The amplification product obtained, containing atleast one fragment SEQ ID N^(o)63, migrates forming a band of 189 basepairs, specific to the genome of the merluccidae. Well 4 is a negativeamplification control.

FIG. 3 represents alignments of nucleotide sequences of 120 base pairs(120 bp) of the gene coding for the cytochrome c oxidase of themitochondrial DNA of different species of fish, in particular gadiformeschosen from the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae. The sequences obtained after sequencingare aligned and compared with the already known sequences. The sequencesof the different species of fish represented are respectively thefollowing (from top to bottom of the figure): Gadus morhua (common orAtlantic cod), Theragra chalcogramma (Alaskan pollock), Melanogrammusaeglefinus (haddock), Merlangius merlangus (whiting), Pollachius virens(pollock), Microgadus tomcod (Atlantic tomcod), Trisopterus luscus(common pout), Coryphaenoides armatus (grenadier), Merluccius capensis(shallow-water Cape hake), Merluccius hubbsi (Argentine hake),Merluccius merluccius (common hake) and Molva molva (ling). The dotsrepresent the bases preserved with those of Gadus morhua (referencesequence).

FIGS. 4-a and 4-b represent an agarose gel coloured with ethidiumbromide on which is deposited a marker measuring 100 bp (M). Thesefigures represent more particularly tests carried out according to thespecific strategy, on different food preparations.

In FIG. 4-a, the DNAs extracted from different food preparations, fillet(well 1), brandade (well 2), cooked dish (well 3), soup (well 4) andrillettes (well 5), were amplified using the primers SEQ ID N^(o)29 andSEQ ID N^(o)32. The amplification product obtained, containing afragment SEQ ID N^(o)64, migrates forming a band of 168 base pairs,specific to Gadus morhua. Well 6 is a negative amplification control.The absence of amplification product for wells 1, 3 and 4 indicates thatthere is no Gadus morhua in the food preparations tested. On the otherhand, the presence of an amplification product in wells 2 and 5indicates the presence of Gadus morhua in the food preparations tested.

In FIG. 4-b, the DNAs extracted from various food preparations, fillet(well 1), brandade (well 2), cooked dish (well 3), soup (well 4) andrillettes (well 5) were amplified using the primers SEQ ID N^(o)41 andSEQ ID N^(o)44. The amplification product obtained, containing afragment SEQ ID N^(o)66, migrates forming a band of 198 base pairs,specific to Theragra chalcogramma. Well 6 is a negative amplificationcontrol. The absence of amplification product for wells 2, 4 and 5indicates that there is no Theragra chalcogramma in the foodpreparations tested. On the other hand the presence of an amplificationproduct in wells 1 and 3 indicates the presence of Theragra chalcogrammain the food preparations tested.

FIGS. 5-a and 5-b represent profiles of crude sequences obtainedaccording to the global strategy.

FIG. 5-a represents more particularly the profile obtained afterextraction of the DNA of a food preparation of the cooked dish type, andamplification using the primers SEQ ID N^(o)2 and SEQ ID N^(o)8. Theamplification product obtained, containing at least one fragment SEQ IDN^(o)58, forms a band of 442 base pairs, specific to the genome of thegadiformes. The sequence obtained at the end of the sequencing is clearand no indetermination appears on the profile. It can therefore bededuced from this that a single species of gadiforme is present in thefood preparation. In the present case, analysis of the sequence revealsthe presence of Gadus morhua in the food preparation.

FIG. 5-b represents more particularly the profile obtained afterextraction of the DNA of a food preparation of the cooked dish type, andamplification using the primers SEQ ID N^(o)2 and SEQ ID N^(o)8. Theamplification product obtained, containing at least one fragment SEQ IDN^(o)58, forms a band of 442 base pairs, specific to the genome of thegadiformes. The sequence obtained at the end of the sequencing presentsnumerous indeterminations (N) which appear on the profile. It cantherefore be deduced from this that the food preparation contains amixture of at least two species of gadiformes. In the present case itwill be necessary, in order to determine precisely the species ofgadiformes present in the food preparation, to carry out a cloning ofthe amplification product obtained, containing at least one fragment SEQID N^(o)58, said cloning allowing separation of the different species ofgadiformes present in the food preparation. At the end of the cloning,it will then be possible to proceed with the sequencing of each of thefragments, and thus to determine the different species present in thefood preparation.

FIG. 6 represents the positions, on the gene coding for the cytochrome coxidase of the mitochondrial DNA, said positions being defined byJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214:

of the oligonucleotide primers of the invention, namely SEQ ID N^(o)1,SEQ ID N^(o)2, SEQ ID N^(o)3, SEQ ID N^(o)4, SEQ ID N^(o)5, SEQ IDN^(o)6, SEQ ID N^(o)7, SEQ ID N^(o)8, SEQ ID N^(o)9, SEQ ID N^(o)10, SEQID N^(o)11, SEQ ID N^(o)12, SEQ ID N^(o)13, SEQ ID N^(o)14, SEQ IDN^(o)15, SEQ ID N^(o)16, SEQ ID N^(o)17, SEQ ID N^(o)18, SEQ ID N^(o)19,SEQ ID N^(o)20, SEQ ID N^(o)21, SEQ ID N^(o)22, SEQ ID N^(o)23, SEQ IDN^(o)24, SEQ ID N^(o)25, SEQ ID N^(o)26, SEQ ID N^(o)27, SEQ ID N^(o)28,SEQ ID N^(o)29, SEQ ID N^(o)30, SEQ ID N^(o)31, SEQ ID N^(o)32, SEQ IDN^(o)33, SEQ ID N^(o)34, SEQ ID N^(o)35, SEQ ID N^(o)36, SEQ ID N^(o)37,SEQ ID N^(o)38, SEQ ID N^(o)39, SEQ ID N^(o)40, SEQ ID N^(o)41, SEQ IDN^(o)42, SEQ ID N^(o)43, SEQ ID N^(o)44, SEQ ID N^(o)45, SEQ ID N^(o)46,SEQ ID N^(o)47, SEQ ID N^(o)48, SEQ ID N^(o)49, SEQ ID N^(o)50, SEQ IDN^(o)51, SEQ ID N^(o)52, SEQ ID N^(o)53, SEQ ID N^(o)54, SEQ ID N^(o)55,SEQ ID N^(o)56, SEQ ID N^(o)57 and,

of the DNA fragments amplified with the help of the oligonucleotideprimers of the invention, namely SEQ ID N^(o)58, SEQ ID N^(o)59, SEQ IDN^(o)60, SEQ ID N^(o)61, SEQ ID N^(o)62, SEQ ID N^(o)63, SEQ ID N^(o)64,SEQ ID N^(o)65, SEQ ID N^(o)66, SEQ ID N^(o)67 and SEQ ID N^(o)68.

FIG. 7 represents the cloning method used within the framework of theinvention in order to separate the different DNA fragments originatingfrom different species of fish in the same mixture.

Part 1 of FIG. 7 represents the pCR®2.1—TOPO plasmid in linear form. Theamplification product of the invention, which contains several copies ofdifferent DNA fragments, is placed in contact with the pCR® 2.1—TOPOplasmid, using a ligase enzyme.

Part 2 represents a part of each of the DNA fragments (initiallycontained in the amplification product) ligated in a pCR®2.1—TOPOplasmid. A plasmid corresponds to each DNA fragment.

Part 3 represents the Escherichia coli (E. coli) bacterial cells.

In part 4, the E. coli bacteria are transformed by introduction of theplasmids represented in part 2. A plasmid corresponds to each E. colibacterium.

Part 5 (symbolized by an arrow) represents the spread of the E. colibacteria on a medium containing an antibiotic (for example ampicillin),in order to select the bacteria that have incorporated a plasmid.

Part 6 represents a dish of gelose on which blue colonies (symbolized by●) and white colonies (symbolized by ◯) have grown. The blue coloniesare characteristic of the bacterial cells in which the DNA fragments arenot ligated to the plasmid, whereas the white colonies arecharacteristic of the bacterial cells in which the DNA fragments areligated with the plasmid.

Part 7 represents the individual sampling of the colonies of whitebacteria (for example 10 bacterial colonies selected at random andsymbolized by the letters A to J) which are individually cultured, as aplasmid, and therefore a specific DNA fragment (symbolized respectivelyby the letters A, B, C, D, E, F, G, H, I and J) corresponds to eachbacterial colony.

In part 8, the bacteria were eliminated in order to recover the plasmidsand their DNA fragments (A to J). Sufficient plasmid DNA is thusobtained in order to sequence it.

Part 9 represents the result of the sequencing of the different DNAfragments A to J, carried out using two primers specific to the plasmidwhich frame the DNA fragment that it is wished to sequence. From the tenfragments sequenced, two different types of sequences are obtained.Thus, the analysed mixture comprises two different species ofgadiformes, in this case Gadus morhua (fragments A, B, D, E, F, G, I andJ), and Merluccius hubbsi (fragments C and H).

Table 1 below represents a list of gadiformes that can be detected andidentified according to the method of the invention. This list is notexhaustive, however; thus, species of gadiformes other than thosespecifically named in table 1 below can be detected and identifiedaccording to the method of the invention. TABLE 1 Families Genus SpeciesCommon name Bregmacerotidae Bregmaceros Bregmaceros sp. unicorn codGadidae Arctogadus Arctogadus glacialis artic cod Boreogadus Boreogadussaida polar cod Brosme Brosme brosme tusk Ciliata Ciliata mustelafive-bearded rockling Eleginus Eleginus gracilis saffron cod Eleginusnavaga wachna cod Enchelyopus Enchelyopus cimbrius four-bearded rocklingGadiculus Gadiculus argenteus silver pollock Gadus Gadus macrocephalusPacific cod Gadus morhua common cod Gadus ogac Greenland codGaidropsarus Gaidropsarus vulgaris common rockling Gaidropsarusmediterraneus three-bearded rockling Lota lota lota burbot MelanogrammusMelanogrammus aeglefinus haddock Merlangius Merlangius merlangus whitingMicrogadus Microgadus proximus tomcod Microgadus tomcod Atlantic tomcodMicromesistius Micromesistius poutassou blue whiting Micromesistiusaustralis Southern whiting Molva Molva dypterygia blue ling Molva molvaling Phycis Phycis blennoides greater forkbeard Phycis chesteri longfinhake Phycis phycis forkbeard Pollachius Pollachius pollachius pollackPollachius virens pollock Raniceps Raniceps raninus tadpole fishTheragra Theragra chalcogramma Alaskan pollock Theragra finnmarchicaNorwegian pollock Trisopterus Trisopterus esmarkii Norway poutTrisopterus luscus common pout Trisopterus capelanus poor codTrisopterus minutus minutus little poor cod Urophycis Urophycisbrasiliensis Brazilian codling Urophycis chuss red hake Urophycisfloridana Southern hake Urophycis regia spotted codling Urophycis tenuiswhite hake Macrouridae Abyssicola Abyssicola macrochir abyssal grenadierAlbatrossia Albatrossia pectoralis giant grenadier Bathygadus Bathygadusmacrops Grenadier sp. Gadomus Gadomus arcuatus Grenadier sp.Coelorinchus Coelorinchus argentatus silver whiptail grenadierCoryphaenoides Coryphaenoides acrolepis Pacific grenadier Coryphaenoidesmexicanus Mexican grenadier Coryphaenoides rupestris roundnose grenadierCynomacrurus Cynomacrurus pirei dogtooth grenadier HymenocephalusHymenocephalus italicus Italian grenadier Lepidorhynchus Lepidorhynchusdenticulatus javelin grenadier Macrous Macrourus berglax grey grenadierMalacocephalus Malacocephalus laevis bearded grenadier MataeocephalusMataeocephalus sturgeon grenadier acipenserinus Nezumia Nezumia aequalissmooth grenadier Sphagemacrurus Sphagemacrurus hirundo graylinggrenadier Trachonurus Trachonurus sulcatus bristly grenadierTrachyrincus Trachyrincus halolepis armourhead grenadier VentrifossaVentrifossa atherodon arrowtooth grenadier Merluccidae MerlucciusMerluccius albidus offshore hake Merluccius australis Southern hakeMerluccius bilinearis silver hake Merluccius capensis shallow-water hakeMerluccius gayi Chilean hake Merluccius hubbsi Argentine hake Merlucciusmerluccius common hake Merluccius paradoxus deep-water Cape hakeMerluccius productus Pacific hake Merluccius senegalensis Senegalesehake Steindachneria Steindachneria argentea silver hake Moridae AntimoraAntimora microlepsis purple antimora Auchenoceros Auchenoceros punctatusahuru Mora Mora moro common mora

The examples below illustrate the invention. They in no way limit it.

EXAMPLE 1 Extraction of DNA

In order to be able to develop a method of detection by geneamplification of biological materials originating from gadiformes inproducts used in agri-foodstuff production, and in all the other fieldsusing organic material, the experiments described in this first examplewere carried out with various types of samples representing potentialsources of the presence of biological materials originating from fish.

1) Extraction of DNA by the Phenol/Chloroform Method

This method is suitable for all the types of samples likely to containorganic material, such as fillet, soup, terrine, pate, fat, flour,fish-based preparations etc.

This method makes use of the technique described in the references HÄNNIet al., 1990, C.R. Acad. Sci. Paris., 310, 365-370 and HÄNNI et al.,1995, Nucl. Acids Res., 23, 881-882, concerning the extraction of DNAfrom bones and teeth.

A quantity of approximately 1 to 2 g of sample of organic material isincubated for two hours at 37° C. in 400 μl of lysis buffer of thefollowing composition:

STE 1× (NaCl 100 mM, Tris 10 mM at pH 7.4, EDTA (ethylenediaminetetraacetic acid) 1 mM),

SDS 2%,

proteinase K at 0.5 mg/ml.

The proteinase K allows the proteins to be degraded and the nucleicacids released. The lysate is then extracted twice with a volume ofphenol/chloroform (1/1). This is centrifuged for 15 minutes at 1000 g,the organic phase is eliminated, which allows the protein part of thelysate to be disposed of. The DNA is precipitated by 1/10 of volume of2M sodium acetate then by 2.5 volumes of isopropanol and centrifuged for30 minutes at 10 000 g. The DNA is taken up in water: a DNA extract isthus obtained. The volume of water is a function of the quantity ofrecovered DNA.

The migration of the DNAs originating from the various samples givesstreaks characteristic of a degraded DNA, which is, however, perfectlyamplifiable (FIG. 1).

2) Extraction of DNA by the Extraction Kit Method

This method is carried out in the conditions described by the supplierQiagen.

EXAMPLE 2 Amplification of DNA

The PCR is carried out with the pair of primers SEQ ID N^(o)2 and SEQ IDN^(o)8 as defined above. The amplifications are realized in a totalvolume of 50 μl containing:

200 μg/ml of BSA (bovine serum albumin),

250 mM of dNTP (deoxynucleotide triphosphate),

300 ng of each primer,

1.5 mM of magnesium chloride (MgCl₂),

PCR 10× (100 mM Tris-HCl pH 8.3; 500 mM KCl) buffer,

1 unit of Taq Polymerase,

qsf 50 μl of sterile distilled water,

1 μl of DNA extract.

The reaction mixture is carried out in a sterile room under ahorizontal-flux hood, in order to avoid contaminations as far aspossible. The PCRs are carried out on an Ependorff PCR apparatus. EachPCR is divided up as follows:

1 initial cycle at a temperature of 94° C. for 2 minutes then,

40 cycles at a temperature of 94° C. for 1 minute, at a temperature of55° C. to 63° C. for 1 minute, at a temperature of 72° C. for 2 minutes;in the last cycle a terminal elongation is carried out at a temperatureof 72° C. for 7 minutes.

The amplification products are analysed by 2% agarose gelelectrophoresis at a constant voltage of 100 V for 30 min, and usingethidium bromide for the visualization of the amplifications obtained.

The PCR reaction using the pair of primers SEQ ID N^(o)2 and SEQ IDN^(o)8 was carried out on a series of DNA extracts from fish. A singleamplification product is observed, containing at least one DNA fragmentSEQ ID N^(o)58 of a length of 442 base pairs (see wells 1 and 2 of FIG.2-a), for the DNAs of gadiformes, in particular chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae.

The PCR reaction using the pair of primers SEQ ID N^(o)5 and SEQ IDN^(o)8 was carried out on a series of DNA extracts from fish. A singleamplification product is observed, containing at least one DNA fragmentSEQ ID N^(o)59 of a length of 328 base pairs (see wells 3 and 4 of FIG.2-a), for the DNAs of gadiformes, in particular chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae (FIG. 2-a).

The PCR reaction using the pair of primers SEQ ID N^(o)20 and SEQ IDN^(o)26 was carried out on a series of DNA extracts from fish. A singleamplification product is observed, containing at least one DNA fragmentSEQ ID N^(o)62 of a length of 318 base pairs (see wells 2 and 3 of FIG.2-b), for the DNAs of gadiformes, in particular chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae.

The PCR reaction using the pair of primers SEQ ID N^(o)23 and SEQ IDN^(o)26 was carried out on a series of DNA extracts from fish. A singleamplification product is observed, containing at least one DNA fragmentSEQ ID N^(o)63 of a length of 189 base pairs (see wells 5 and 6 of FIG.2-b), for the DNAs of gadiformes, in particular chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae (FIG. 2-b).

The whole of the PCR product (amplification product) can be purifieddirectly using the “QIAquick PCR Purification Kit” kit from Qiagenaccording to the stated conditions. The whole of the PCR product ispassed over a column of silica gel. The nucleic acids are retained onthe column whereas the other residues of the PCR are eluted bymicrocentrifugation. The impurities are eliminated and the DNA is elutedwith Tris buffer or water. The thus-purified DNA is visualized andquantified on 2% agarose gel.

EXAMPLE 3 Characterization of the DNA Fragments by AmplificationFollowed by a Direct Sequencing (“Global Strategy”)

The automatic sequencing is carried out on the purified PCR products.

The primers used are those which served to amplify the DNA fragment orfragments that it is sought to characterize. The “Perkin-Elmer” kit isused for the PCR reaction, which is carried out over 25 cycles in theconditions stated by the supplier. Each amplification product obtainedat the end of the PCR reaction is precipitated with ethanol anddeposited on a polyacrylamide gel. The sequences obtained are thencompared with those of the banks or with the sequences already obtained(FIG. 3).

EXAMPLE 4 Characterization of the DNA Fragments by Direct Amplification(“Specific Strategy”)

The PCR is carried out with the pairs of primers (SEQ ID N^(o)29 and SEQID N^(o)32) and (SEQ ID N^(o)41 and SEQ ID N^(o)44).

The amplifications are carried out in a total volume of 50 μlcontaining:

200 μg/ml of BSA,

250 mM of dNTP,

300 ng of each primer,

1.5 mM of MgCl₂,

PCR 10× (100 mM Tris-HCl pH 8.3; 500 mM KCl) buffer,

1 unit of Taq Polymerase,

qsf 50 μl of sterile distilled water,

1 μl of DNA extract

The reaction mixture is carried out in a sterile room under ahorizontal-flux hood, in order to avoid contaminations as far aspossible. The PCRs are carried out on an Ependorff PCR apparatus.

Each PCR is divided up as follows:

1 initial cycle at a temperature of 94° C. for 2 minutes then,

40 cycles at a temperature of 94° C. for 1 minute, at a temperature ofapproximately 60° C. to approximately 65° C. for 1 minute, at atemperature of 72° C. for 2 minutes; in the last cycle a terminalelongation is carried out at a temperature of 72° C. for 7 minutes.

The amplification product is analysed by 2% agarose gel electrophoresisat a constant voltage of 100 V for 30 min, and using ethidium bromidefor the visualization of the amplification obtained (FIG. 4).

The use of the primers SEQ ID N^(o)29 and SEQ ID N^(o)32 allows anamplification product to be obtained containing a DNA fragment SEQ IDN^(o)64 which migrates forming a band of 168 base pairs, specific toGadus morhua (FIG. 4-a).

The use of the primers SEQ ID N^(o)41 and SEQ ID N^(o)44 allows anamplification product to be obtained containing a DNA fragment SEQ IDN^(o)66 which migrates forming a band of 198 base pairs, specific toTheragra chalcogramma (FIG. 4-b).

EXAMPLE 5 Characterization of a Food Preparation Containing a Mixture ofat Least Two Different Species of Gadiformes

According to an advantageous embodiment, the present invention allowsthe detection and identification of a mixture containing at least twodifferent species of gadiformes. It is in fact common to prepare foodpreparations containing several species of fish (cooked dishes, soup,pates, terrine etc.). Thus, it may happen in particular that foodpreparations are constituted by a mixture of species containing aspecies of fish that is of less economic value compared with anotherspecies which should have been the only one found in the preparation(example: brandade of cod prepared with a little common or Atlantic cod(Gadus morhua), and a lot of Pacific cod (Gadus macrocephalus) which isless exploited and less expensive).

1) Global Strategy

When the sample of organic material to be analysed comprises a mixtureof different species of fish, the amplification product obtained at theend of the PCR reaction contains several copies of different DNAfragments or sequences. However, when a sequencing of such anamplification product is carried out, a sequence is obtained whichcontains numerous indeterminations: it is therefore impossible todetermine what species of fish are present in the sample of organicmaterial to be analysed. According to an advantageous embodiment of theinvention, a cloning of the amplification product obtained is thencarried out: it is then possible to proceed with the sequencing of thedifferent amplified DNA fragments thus separated from one another usingthe cloning.

The extraction and the amplification of the DNA contained in a sample oforganic material containing a mixture of different species of fish iscarried out in the way described above (see examples 1 and 2 above). Atthe end of the PCR reaction, a single amplification product is observed(containing at least one DNA fragment represented by SEQ ID N^(o)58 of alength of 442 base pairs), obtained using the primer pair (SEQ IDN^(o)2, SEQ ID N^(o)8) allowing amplification of all the species ofgadiformes.

Said amplification product is therefore able to contain different DNAfragments characteristic of different species of gadiformes. In thepresent example, the amplification product (containing at least one DNAfragment represented by SEQ ID N^(o)58) contains different DNA fragmentswhich it is sought to separate in order to be able to identify them.According to an advantageous embodiment of the invention, the use of acloning method allows the separation of all the different DNA fragmentscontained in the amplification product, and thus the identification ofeach of these fragments (FIG. 7).

After purification of the amplification product (containing at least oneDNA fragment represented by SEQ ID N^(o)58), this latter is cloned usingthe “TOPO TA cloning kit” system from Invitrogen according to the statedconditions, in order to isolate and obtain numerous identical copies ofall the different DNA fragments contained in the amplification product.For this purpose, the amplification product is inserted by ligation inthe “pCR®2.1—TOPO® 3.9 kb” plasmid marketed by Invitrogen, after cuttingof said plasmid using a restriction enzyme: EcoR I (FIG. 7-1). Each DNAfragment contained in the amplification product will be inserted in a“pCR®2.1—TOPO®” plasmid (FIG. 7-2). A plasmid therefore corresponds toeach DNA fragment. All the DNA fragments contained in the amplificationproduct SEQ ID N^(o)58 are thus separated during this stage.

After ligation of each of the DNA fragments in a plasmid, each plasmidis introduced into a bacterium, for example Escherichia coli (E. coli)(FIG. 7-4), by a method called “transformation” which involves anosmotic shock and a temperature shock or electric shock. The E. colibacteria thus obtained are cultured on gelose in order to be multiplied,in the presence of an antibiotic (for example ampicillin). The presenceof the antibiotic allows the E. coli bacteria that have incorporated aplasmid to be selected, as the plasmids naturally possess a generesistant to an antibiotic. Thus, the E. coli bacteria that have notincorporated a plasmid will not be able to develop.

A visual system is used to select the E. coli bacteria that haveincorporated a plasmid in which a DNA fragment is ligated. In fact, tworeagents (IPTG and X-Gal) were introduced into the gelose-containingmedium which, on contact with the β-galactosidase enzyme, produce a bluecolouring. The colonies of blue bacteria obtained are those whichsynthesize β-galactosidase, and which contain a plasmid in which a DNAfragment is not ligated. The colonies of white bacteria obtained arethose which do not synthesize β-galactosidase, and which contain aplasmid in which a DNA fragment is ligated. This is linked with the factthat the DNA fragment is inserted in the plasmid by means of a genecoding for the β-galactosidase enzyme whose synthesis it blocks.

Each white bacterial colony obtained contains a single amplified DNAfragment or sequence, said DNA fragment or sequence corresponding to oneand the same species of gadiformes. The plasmid DNAs containing the DNAfragments must then be recovered, i.e. separated from the bacterial DNAsin order to be sequenced. According to an advantageous embodiment of themethod of the invention, some ten or so plasmid DNAs (symbolizedrespectively by the letters A, B, C, D, E, F, G, H, I and J on FIG. 7-8)are recovered, originating from 10 independent white bacterial coloniesselected at random (represented by the letters A to J on FIG. 7-7). Abacterial culture is prepared from each white colony selected. Thebacterial DNA is eliminated using the “QIAprep Spin Miniprep Kit” systemfrom Qiagen according to the stated conditions. This purification aimsessentially to eliminate the traces of the remaining bacterial DNA. Itis carried out on resin which fixes the small DNA molecules (such asthose of plasmid DNA), and not the bacterial DNA which is much longer.The plasmid DNA is fixed on the resin then eluted.

The 10 plasmid DNAs (symbolized by the letters A to J on FIG. 7-8) thusrecovered are then sequenced with the primers supplied in the Invitrogenkit. The 10 sequences obtained are analysed so as to identify whetherthe profiles are different or not. The results obtained (FIG. 7-9)indicate that the sample of organic material comprises a mixture of twospecies, in this case Gadus morhua (fragments A, B, D, E, F, G, I and J)which is a fish belonging to the family of the gadidae, and Merlucciushubbsi (fragments C and H) which is a fish belonging to the family ofthe merluccidae.

Thus, if the DNA extracted from the sample of organic material to beanalysed contains only a single species of gadiformes, a single sequenceprofile will be obtained for the 10 sampled and sequenced clones. Thecomparison of the obtained sequence with the reference sequences willallow identification of the species present in the sample of organicmaterial.

If, on the other hand, the DNA extracted from the sample of organicmaterial to be analysed is representative of several species ofgadiformes, different profiles will be obtained correspondingrespectively to the number of species present in the sample. Thecomparison of the different obtained sequences with the referencestandards will allow identification respectively of the differentspecies present in the sample of organic material.

It is important to note that the number of white bacterial coloniessampled can be increased if it is suspected that a large number ofspecies are present in the sample of organic material to be analysed.

a) Detection and identification of a mixture of gadiformes: the Alaskanpollock (Theragra chalcogramma) belonging to the family of the gadidae,and the Argentine hake (Merluccius hubbsi) belonging to the family ofthe merluccidae.

The extraction of the DNA from a fish-based sample is carried out in theway described in Example 1 above.

The PCR is carried out with the primers SEQ ID N^(o)5 and SEQ ID N^(o)8as defined above. A single amplification product is observed, containingat least one DNA fragment represented by SEQ ID N^(o)59 of a length of328 base pairs, characteristic of DNA of gadiformes, in particularchosen from the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae.

Said amplification product is purified, cloned then sequenced in the waydescribed above. Two sequence profiles are observed on the 10 clonesanalysed. The interpretation of these two profiles shows that theycorrespond respectively to one species of gadidae: the Alaskan pollock(Theragra chalcogramma), and one species of merluccidae: the Argentinehake (Merluccius hubbsi).

b) Detection and identification of a mixture of gadidae: the Atlanticcod (Gadus morhua) and the ling (Molva molva).

The extraction of the DNA from a fish-based sample is carried out in theway described in Example 1 above.

The PCR is carried out with the primers SEQ ID N^(o)14 and SEQ IDN^(o)17 as defined above. A single amplification product is observed,containing at least one DNA fragment represented by SEQ ID N^(o)61 of alength of 237 base pairs, characteristic of DNA of gadidae.

Said amplification product is purified, cloned then sequenced in the waydescribed above. Two sequence profiles are observed on the 10 clonesanalysed. The interpretation of these two profiles shows that theycorrespond respectively to two different species of gadidae: theAtlantic cod (Gadus morhua) and the ling (Molva molva).

c) Detection and identification of a mixture of merluccidae: the Capehake (Merluccius capensis) and the common hake (Merluccius merluccius).

The extraction of the DNA from a fish-based sample is carried out in theway described in Example 1 above.

The PCR is carried out with the primers SEQ ID N^(o)23 and SEQ IDN^(o)26 as defined above. A single amplification product is observed,containing at least one DNA fragment represented by SEQ ID N^(o)63 of alength of 189 base pairs, characteristic of DNA of merluccidae.

Said amplification product is purified, cloned then sequenced in the waydefined above. Two sequence profiles are observed on the 10 clonesanalysed. The interpretation of these two profiles shows that theycorrespond respectively to two different species of merluccidae: theCape hake (Merluccius capensis) and the common hake (Merlucciusmerluccius).

2) Specific Strategy

In the case of the specific strategy, it is also possible to detect andidentify a mixture of different species, without using cloning, sinceprimers specific to 5 different species of gadidae are available. It isthus possible to detect and identify a mixture between precisely these 5species of gadidae.

The example below illustrates the detection and the identification of amixture of two species of gadidae: the Atlantic cod (Gadus morhua) andthe Alaskan pollock (Theragra chalcogramma).

The extraction of the DNA from a fish-based sample is carried out in theway described in Example 1 above.

The PCR is carried out with successively the 5 pairs of primers asdefined above for detection and direct identification, namely:

(SEQ ID N^(o)29 and SEQ ID N^(o)32),

(SEQ ID N^(o)35 and SEQ ID N^(o)38),

(SEQ ID N^(o)41 and SEQ ID N^(o)44),

(SEQ ID N^(o)47 and SEQ ID N^(o)50) and,

(SEQ ID N^(o)53 and SEQ ID N^(o)56).

Two amplification products are observed, containing respectively a DNAfragment represented by SEQ ID N^(o) 64 of a length of 168 base pairs,and a DNA fragment represented by SEQ ID N^(o) 66 of a length of 198base pairs, characteristic of DNA of gadidae. On the other hand noamplification band is observed for the other wells (corresponding to theother primers).

Thus, it is deduced from this that the sample of organic material to beanalysed contains Atlantic cod (Gadus morhua) and Alaskan pollock(Theragra chalcogramma).

1. Method for detecting the presence of biological materials originatingfrom gadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, in a sample of organicmaterial, characterized in that the presence of mitochondrial DNAoriginating from gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae in saidorganic material is determined by amplification of at least one sequenceor fragment of mitochondrial DNA specific to the genome of thegadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, and contained in themitochondrial DNA extracted from said sample, namely at least onesequence or fragment present in the genomes of the gadiformes chosenfrom the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, but absent from the genomes of the otheranimal genera, and in particular of the other animal species.
 2. Methodfor detecting the presence of biological materials originating fromgadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, in a sample of organicmaterial, and for identifying the genus, in particular of at least onespecies of gadiformes chosen from the group constituted by the gadidae,the merluccidae, the macrouridae and/or the moridae, present in saidsample, characterized in that the presence of mitochondrial DNAoriginating from gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae in saidorganic material is determined by amplification of at least one sequenceor fragment of mitochondrial DNA specific to the genome of thegadiformes chosen from the group constituted by the gadidae, themerluccidae, the macrouridae and/or the moridae, and contained in themitochondrial DNA extracted from said sample, namely at least onesequence or fragment present in the genomes of the gadiformes chosenfrom the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, but absent from the genomes of the otheranimal genera, in particular of the other animal species, and in that atleast one sequence or fragment of mitochondrial DNA specific to thegenome of the gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae, thusamplified, is compared with other mitochondrial DNA sequences of thegenome of the gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae, saidsequence of mitochondrial DNA or said fragment of mitochondrial DNAspecific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae, thus amplified, displaying at least approximately 50% identity,in particular approximately 60% identity with the other aforementionedsequences of mitochondrial DNA of the genome of the gadiformes chosenfrom the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae.
 3. Method according to claim 1,characterized in that it allows the detection and optionally theidentification of the presence of gadidae, in particular chosen from thegroup constituted by Gadus morhua (common cod), Melanogrammus aeglefinus(haddock), Merlangius merlangus (whiting), Micromesistius poutassou(blue whiting), Pollachius virens (pollock), Pollachius pollachius(pollack), Trisopterus luscus (common pout), Trisopterus minutuscapelanus (poor cod), Theragra chalcogramma (Alaskan pollock), Bromebrome (tusk), Molva molva (ling) or Molva dypterygia dypterigia (blueling).
 4. Method according to claim 1, characterized in that it allowsthe detection and optionally the identification of the presence ofmerluccidae, in particular chosen from the group constituted byMerluccius albidus (offshore hake), Merluccius australis (Southernhake), Merluccius bilinearis (silver hake), Merluccius capensis(shallow-water Cape hake), Merluccius gayi (Chilean hake), Merlucciushubbsi (Argentine hake), Merluccius merluccius (common hake), Merlucciusparadoxus (deep-water Cape hake), Merluccius productus (North Pacifichake), Merluccius senegalensis (Senegalese hake), Steindachneriaargentea (silver hake).
 5. Method according to claim 1, characterized inthat the amplified sequence or fragment of the genome of the gadiformeschosen from the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, is situated in the central part of thegene coding for the cytochrome c oxidase of the mitochondrial DNA,delimited by the nucleotides situated in the vicinity of positions 6100and 6601, and in particular in the vicinity of positions 6120 and 6590,and preferably in the vicinity of positions 6131 and 6580 of the genecoding for the cytochrome c oxidase of the mitochondrial DNA. 6.Oligonucleotides characterized in that that they are chosen from those:(1)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)1 (positions 6131 to6154 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TRT AYC ARC AYY TRT TYT GRT TCT K

in which R is A or G, Y is C or T, K is G or T, on condition that thefollowing sequences are excluded: CGG GAT CCT GTT CTG ATT CTT GAT TTC C(SEQ ID NO: 69) and CGA CGG GAT CCC AAC ACC TGT TTC GAT CAT CGC GGC AAC(SEQ ID NO: 70) or comprising the following sequence SEQ ID N^(o)2(positions 6134 to 6154 according to Johansen and Bakke, 1996, MolecularMarine Biology and Biotechnology, 5(3) 203-214): AYC ARC AYY TRT TYT GRTTCT

in which Y is C or T, R is A or G, or constituted by the followingsequence SEQ ID N^(o)3 (positions 6139 to 6153 according to Johansen andBakke, 1996, Molecular Marine Biology and Biotechnology, 5(3) 203-214):AYY TRT TYT GRT TCT

in which Y is C or T, R is A or G, or those (2)—displaying a sequenceidentity of at least 80%, preferably 90% and advantageously 95% with anoligonucleotide constituted by a sequence of approximately 15 to 25nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)4 (positions 6244 to 6269 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): TTY GGN YAY ATR GGN ATR GTN TGA GC

in which Y is C or T, N is A, C, G or T, R is A or G, or comprising thefollowing sequence SEQ ID N^(o)5 (positions 6247 to 6269 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): GGN YAY ATR GGN ATR GTN TGA GC

in which N is A, C, G or T, Y is C or T, R is A or G, or constituted bythe following sequence SEQ ID N^(o)6 (positions 6253 to 6269 accordingto Johansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): RGG NAT RGT NTG AGC

in which R is A or G, N is A, C, G or T, or those (3)—displaying asequence identity of at least 80%, preferably 90% and advantageously 95%with an oligonucleotide constituted by a sequence of approximately 15 to25 nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)7 (positions 6556 to 6580 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): TAY GTW GTN GCN CAY TTY CAC TAC G

in which Y is C or T, W is A or T, N is A, C, G or T, or comprising thefollowing sequence SEQ ID N^(o)8 (positions 6556 to 6575 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): TAY GTW GTN GCN CAY TTY CA

in which Y is C or T, W is A or T, N is A, C, G or T, or constituted bythe following sequence SEQ ID N^(o)9 (positions 6556 to 6570 accordingto Johansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): TAY GTW GTN GCN CAY

in which Y is C or T, W is A or T, N is A, C, G or T, or those(4)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)10 (positions 6131 to6154 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TRT AYC ARC AYY TRT TYT GRT TCT K

in which R is A or G, Y is C or T, K is G or T, or comprising thefollowing sequence SEQ ID N^(o)11 (positions 6134 to 6154 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): ACC AAC ACT TAT TCT GAT TCT

or constituted by the following sequence SEQ ID N^(o)12 (positions 6139to 6154 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): AYY AAC ACT TAT TCT

in which Y is C or T, or those (5)—displaying a sequence identity of atleast 80%, preferably 90% and advantageously 95% with an oligonucleotideconstituted by a sequence of approximately 15 to 25 nucleotides, inparticular 20 to 25 nucleotides, comprised in the following sequence SEQID N^(o)13 (positions 6277 to 6303 according to Johansen and Bakke,1996, Molecular Marine Biology and Biotechnology, 5(3) 203-214): CYA TYGGMC TYT YGG YTT TAT YGT V

in which Y is C or T, M is A or C, V is A, C or G, or comprising thefollowing sequence SEQ ID N^(o)14 (positions 6283 to 6303 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): GGC CTC CTT GGC TTT ATT GTA

or constituted by the following sequence SEQ ID N^(o)15 (positions 6288to 6303 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): YTY GGY TTT ATT GTV

in which Y is C or T, V is A, C or G, or those (6)—displaying a sequenceidentity of at least 80%, preferably 90% and advantageously 95% with anoligonucleotide constituted by a sequence of approximately 15 to 25nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)16 (positions 6496 to 6522 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): GGM YTW ACA GGN ATY RTH YTR GCY AA

in which M is A or C, W is A or T, Y is C or T, N is A, C, G or T, R isA or G, H is A, C or T, or comprising the following sequence SEQ IDN^(o)17 (positions 6496 to 6519 according to Johansen and Bakke, 1996,Molecular Marine Biology and Biotechnology, 5(3) 203-214): GGC TTA ACAGGA ATT GTA CTA GCT

or constituted by the following sequence SEQ ID N^(o)18 (positions 6496to 6510 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GGC TTA ACA GGA ATT

or those (7)—displaying a sequence identity of at least 80%, preferably90% and advantageously 95% with an oligonucleotide constituted by asequence of approximately 15 to 25 nucleotides, in particular 20 to 25nucleotides, comprised in the following sequence SEQ ID N^(o)19(positions 6195 to 6219 according to Johansen and Bakke, 1996, MolecularMarine Biology and Biotechnology, 5(3) 203-214): CGG RAT AAT YTC YCA YATYGT AGC C

in which R is A or G, Y is C or T, or comprising the following sequenceSEQ ID N^(o)20 (positions 6200 to 6219 according to Johansen and Bakke,1996, Molecular Marine Biology and Biotechnology, 5(3) 203-214): TAA TYTCYC AYA TYG TAG CC

in which Y is C or T, or constituted by the following sequence SEQ IDN^(o)21 (positions 6205 to 6219 according to Johansen and Bakke, 1996,Molecular Marine Biology and Biotechnology, 5(3) 203-214): TCY CAY ATYGTA GCC

in which Y is C or T, or those (8)—displaying a sequence identity of atleast 80%, preferably 90% and advantageously 95% with an oligonucleotideconstituted by a sequence of approximately 15 to 25 nucleotides, inparticular 20 to 25 nucleotides, comprised in the following sequence SEQID N^(o)22 (positions 6324 to 6348 according to Johansen and Bakke,1996, Molecular Marine Biology and Biotechnology, 5(3) 203-214): AGT BGGRAT RGA YGT DGA YAC MCG T

in which B is C, G or T, R is A or G, Y is C or T, D is A, G or T, M isA or C, or comprising the following sequence SEQ ID N^(o)23 (positions6329 to 6348 according to Johansen and Bakke, 1996, Molecular MarineBiology and Biotechnology, 5(3) 203-214): GRA TRG AYG TDG AYA CMC GT

in which R is A or G, Y is C or T, D is A, G or T, M is A or C, orconstituted by the following sequence SEQ ID N^(o)24 (positions 6334 to6348 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GAY GTD GAY ACM CGT

in which Y is C or T, D is A, G or T, M is A or C, or those(9)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)25 (positions 6498 to6523 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): ACT TAC AGG NAT YRT HCT RGC YAA YT

in which N is A, C, G or T, Y is C or T, R is A or G, H is A, C or T, orcomprising the following sequence SEQ ID N^(o)26 (positions 6498 to 6517according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): ACT TAC AGG NAT YRT HCT RG

in which N is A, C, G or T, Y is C or T, R is A or G, H is A, C or T, orconstituted by the following sequence SEQ ID N^(o)27 (positions 6498 to6512 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): ACT TAC AGG NAT YRT

in which N is A, C, G or T, Y is C or T, R is A or G, or those(10)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)28 (positions 6399 to6423 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): AGT YTT YAG YTG AYT AGC AAC YYT V

in which Y is C or T, V is A, C or G, or comprising the followingsequence SEQ ID N^(o)29 (positions 6404 to 6423 according to Johansenand Bakke, 1996, Molecular Marine Biology and Biotechnology, 5(3)203-214): TTA GCT GAT TAG CAA CTT TA

or constituted by the following sequence SEQ ID N^(o)30 (positions 6409to 6423 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TGA YTA GCA ACY YTV

in which Y is C or T, V is A, C or G, or those (11)—displaying asequence identity of at least 80%, preferably 90% and advantageously 95%with an oligonucleotide constituted by a sequence of approximately 15 to25 nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)31 (positions 6552 to 6577 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): RTA YTA YGT AGT MGC YCA YTT YCA CT

in which R is A or G, Y is C or T, M is A or C, or comprising thefollowing sequence SEQ ID N^(o)32 (positions 6552 to 6572 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): GTA TTA CGT AGT AGC CCA TT

or constituted by the following sequence SEQ ID N^(o)33 (positions 6552to 6566 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): RTA YTA YGT AGT MGC

in which R is A or G, Y is C or T, M is A or C, or those (12)—displayinga sequence identity of at least 80%, preferably 90% and advantageously95% with an oligonucleotide constituted by a sequence of approximately15 to 25 nucleotides, in particular 20 to 25 nucleotides, comprised inthe following sequence SEQ ID N^(o)34 (positions 6237 to 6261 accordingto Johansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): AGA RCC NTT YGG RYA YAT RGG HAT R

in which R is A or G, N is A, C, G or T, Y is C or T, H is A, C or T, orcomprising the following sequence SEQ ID N^(o)35 (positions 6242 to 6261according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): CCT TTG GAT ATA TAG GCA TG

or constituted by the following sequence SEQ ID N^(o)36 (positions 6248to 6261 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GGR YAY ATR GGH ATR

in which R is A or G, Y is C or T, H is A, C or T, or those(13)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)37 (positions 6381 to6406 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): YAT YCC RAC AGG YGT WAA AGT YTT YA

in which Y is C or T, R is A or G, W is A or T, or comprising thefollowing sequence SEQ ID N^(o)38 (positions 6381 to 6400 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): TAT CCC AAC AGG TGT AAA AG

or constituted by the following sequence SEQ ID N^(o)39 (positions 6381to 6395 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TAT YCC RAC AGG YGT

in which Y is C or T, R is A or G, or those (14)—displaying a sequenceidentity of at least 80%, preferably 90% and advantageously 95% with anoligonucleotide constituted by a sequence of approximately 15 to 25nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)40 (positions 6267 to 6291 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): AGC YAT RAT RGC YAT YGG MCT YCT Y

in which Y is C or T, R is A or G, M is A or C, or comprising thefollowing sequence SEQ ID N^(o)41 (positions 6272 to 6291 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): TGA TGG CTA TTG GCC TCC TC

or constituted by the following sequence SEQ ID N^(o)42 (positions 6277to 6291 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): GCY ATY GGM CTY CTY

in which Y is C or T, M is A or C, or those (15)—displaying a sequenceidentity of at least 80%, preferably 90% and advantageously 95% with anoligonucleotide constituted by a sequence of approximately 15 to 25nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)43 (positions 6451 to 6475 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): HCC BMT MCT BTG RGC CCT V GG YTT YA

in which H is A, C or T, B is C, G or T, M is A or C, R is A or G, V isA, C or G, Y is C or T, or comprising the following sequence SEQ IDN^(o)44 (positions 6451 to 6469 according to Johansen and Bakke, 1996,Molecular Marine Biology and Biotechnology, 5(3) 203-214): CCC TCT ACTCTG AGC CCT AG

or constituted by the following sequence SEQ ID N^(o)45 (positions 6451to 6464 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): HCC BMT MCT BTG RGC

in which H is A, C or T, B is C, G or T, M is A or C, R is A or G, orthose (16)—displaying a sequence identity of at least 80%, preferably90% and advantageously 95% with an oligonucleotide constituted by asequence of approximately 15 to 25 nucleotides, in particular 20 to 25nucleotides, comprised in the following sequence SEQ ID N^(o)46(positions 6194 to 6219 according to Johansen and Bakke, 1996, MolecularMarine Biology and Biotechnology, 5(3) 203-214): TSG RAT AAT YTC YCA YATYGT AGC V

in which S is C or G, R is A or G, Y is C or T, V is A, C or G, orcomprising the following sequence SEQ ID N^(o)47 (positions 6200 to 6219according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAA TTT CTC ACA TCG TAG CG

or constituted by the following sequence SEQ ID N^(o)48 (positions 6205to 6219 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): TCY CAY ATY GTA GCV

in which Y is C or T, V is A, C or G, or those (17)—displaying asequence identity of at least 80%, preferably 90% and advantageously 95%with an oligonucleotide constituted by a sequence of approximately 15 to25 nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)49 (positions 6342 to 6366 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): YAC MCG WGC HTA CTT YAC ATC YGC A

in which Y is C or T, M is A or C, W is A or T, H is A, C or T orcomprising the following sequence SEQ ID N^(o)50 (positions 6342 to 6361according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TAC ACG TGC CTA CTT TAC AT

or constituted by the following sequence SEQ ID N^(o)51 (positions 6342to 6356 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): YAC MCG WGC HTA CTT

in which Y is C or T, M is A or C, W is A or T, H is A, C or T, or those(18)—displaying a sequence identity of at least 80%, preferably 90% andadvantageously 95% with an oligonucleotide constituted by a sequence ofapproximately 15 to 25 nucleotides, in particular 20 to 25 nucleotides,comprised in the following sequence SEQ ID N^(o)52 (positions 6152 to6177 according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): TCT KCG GNC AYC CYG AAG THT AYA TH

in which K is G or T, N is A, C, G or T, Y is C or T, H is A, C or T, orcomprising the following sequence SEQ ID N^(o)53 (position 6158 to 6177according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GAC ACC CCG AAG TAT ACA TA

or constituted by the following sequence SEQ ID N^(o)54 (positions 6163to 6177 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): CCY GAA GTH TAY ATH

in which Y is C or T, H is A, C or T, or those (19)—displaying asequence identity of at least 80%, preferably 90% and advantageously 95%with an oligonucleotide constituted by a sequence of approximately 15 to25 nucleotides, in particular 20 to 25 nucleotides, comprised in thefollowing sequence SEQ ID N^(o)55 (positions 6303 to 6328 according toJohansen and Bakke, 1996, Molecular Marine Biology and Biotechnology,5(3) 203-214): VTG RGC YCA YCA CAT RTT YAC AGT BG

in which V is A, C or G, R is A or G, Y is C or T, B is C, G or T, orcomprising the following sequence SEQ ID N^(o)56 (positions 6303 to 6322according to Johansen and Bakke, 1996, Molecular Marine Biology andBiotechnology, 5(3) 203-214): GTG AGC CCA TCA CAT GTT TA

or constituted by the following sequence SEQ ID N^(o)57 (positions 6303to 6317 according to Johansen and Bakke, 1996, Molecular Marine Biologyand Biotechnology, 5(3) 203-214): VTG RGC YCA YCA CAT,

in which V is A, C or G, R is A or G, Y is C or T.
 7. Pairs of primerscharacterized in that that they are constituted: by any one of theoligonucleotides SEQ ID N^(o)1, SEQ ID N^(o)2, SEQ ID N^(o)3, SEQ IDN^(o)4, SEQ ID N^(o)5, SEQ ID N^(o)6, and any one of theoligonucleotides SEQ ID N^(o)7, SEQ ID N^(o)8, SEQ ID N^(o)9 accordingto claim 6 or, by any one of the oligonucleotides SEQ ID N^(o)10, SEQ IDN^(o)11, SEQ ID N^(o)12, SEQ ID N^(o)13, SEQ ID N^(o)14, SEQ ID N^(o)15,and any one of the oligonucleotides SEQ ID N^(o)16, SEQ ID N^(o)17, SEQID N^(o)18 according to claim 6 or, by any one of the oligonucleotidesSEQ ID N^(o)19, SEQ ID N^(o)20, SEQ ID N^(o)21, SEQ ID N^(o)22, SEQ IDN^(o)23, SEQ ID N^(o)24, and any one of the oligonucleotides SEQ IDN^(o)25, SEQ ID N^(o)26, SEQ ID N^(o)27 according to claim 6 or, by anyone of the oligonucleotides SEQ ID N^(o)28, SEQ ID N^(o)29, SEQ IDN^(o)30, and any one of the oligonucleotides SEQ ID N^(o)31, SEQ IDN^(o)32, SEQ ID N^(o)33 according to claim 6 or, by any one of theoligonucleotides SEQ ID N^(o)34, SEQ ID N^(o)35, SEQ ID N^(o)36, and anyone of the oligonucleotides SEQ ID N^(o)37, SEQ ID N^(o)38, SEQ IDN^(o)39 according to claim 6 or, by any one of the oligonucleotides SEQID N^(o)40, SEQ ID N^(o)41, SEQ ID N^(o)42, and any one of theoligonucleotides SEQ ID N^(o)43, SEQ ID N^(o)44, SEQ ID N^(o)45according to claim 6 or, by any one of the oligonucleotides SEQ IDN^(o)46, SEQ ID N^(o)47, SEQ ID N^(o)48, and any one of theoligonucleotides SEQ ID N^(o)49, SEQ ID N^(o)50, SEQ ID N^(o)51according to claim 6 or, by any one of the oligonucleotides SEQ IDN^(o)52, SEQ ID N^(o)53, SEQ ID N^(o)54, and any one of theoligonucleotides SEQ ID N^(o)55, SEQ ID N^(o)56, SEQ ID N^(o)57according to claim 6, and advantageously constituted by the pair ofoligonucleotides chosen from the following pairs: (SEQ ID N^(o)2 and SEQID N^(o)8), (SEQ ID N^(o)5 and SEQ ID N^(o)8), (SEQ ID N^(o)11 and SEQID N^(o)17), (SEQ ID N^(o)14 and SEQ ID N^(o)17), (SEQ ID N^(o)20 andSEQ ID N^(o)26), (SEQ ID N^(o)23 and SEQ ID N^(o)26), (SEQ ID N^(o)29and SEQ ID N^(o)32), (SEQ ID N^(o)35 and SEQ ID N^(o)38), (SEQ IDN^(o)41 and SEQ ID N^(o)44), (SEQ ID N^(o)47 and SEQ ID N^(o)50), (SEQID N^(o)53 and SEQ ID N^(o)56).
 8. Method according to claim 1,characterized in that the amplification of at least one sequence orfragment of mitochondrial DNA specific to the genome of the gadiformeschosen from the group constituted by the gadidae, the merluccidae, themacrouridae and/or the moridae, is carried out by the polymerase chainamplification method (PCR), comprising a repetition of the cycle of thefollowing stages: heating of the DNA extracted from the sample oforganic material, so as to separate the DNA into two single-chainstrands, hybridization of oligonucleotide primers according to claims 6and 7 or sequences CGG GAT CCT GTT CTG ATT CTT GAT TTC C (SEQ ID NO: 69)and CGA CGG GAT CCC AAC ACC TGT TTC GAT CAT CGC GGC AAC (SEQ ID NO: 70),to the monocatenary DNA strands at an adequate temperature, andelongation of the oligonucleotide primers by a polymerase at an adequatetemperature, in order to obtain at least one amplified DNA sequence orfragment specific to the genome of the gadiformes chosen from the groupconstituted by the gadidae, the merluccidae, the macrouridae and/or themoridae.
 9. Method according to claim 1, characterized in that theobtained amplified mitochondrial DNA fragment(s) contained in theamplification product is (are) identified: by sequencing of at least oneamplified DNA fragment, and in particular of one amplified DNA fragmentor, directly, by visualization of the presence of the amplificationproduct by gel electrophoresis.
 10. Method according to claim 9,characterized in that the sequencing of each of the amplified DNAfragments is preceded by a cloning method when the sample of organicmaterial comprises a mixture of different DNA fragments originating fromdifferent species of gadiformes chosen from the group constituted by thegadidae, the merluccidae, the macrouridae and/or the moridae, saidcloning method permitting the separation from said mixture of thedifferent DNA fragments originating from the different species ofgadiformes.
 11. Method according to claim 1, characterized in that theDNA extracted from the sample of organic material is: non-degraded DNAoriginating in particular from a fresh sample or, degraded DNA,originating in particular from a sample that has been transformed, inparticular cooked, lyophilized, dried, pickled, appertized, pasteurizedetc.
 12. DNA fragment as amplified at the end of the method according toclaim 1, characterized in that it comprises approximately 100 toapproximately 500 base pairs.
 13. DNA fragment according to claim 12,characterized in that it displays a sequence identity of at least 80%,preferably 90% and advantageously 95% with at least one of the sequencescontained in: the following SEQ ID N^(o)58: AYCARCAYYT RTTCTGATTCTKCGGNCAYC CYGAAGTHTA YATHCTNATY YTMCCHGGMT TCGGRATAAT YTCYCAYATYGTAGCVTAYT AYTCAGGNAA RMAAGARCCN TTYGGRYAYA TRGGHATRGT NTGAGCYATRATRGCYATYG GMCTYCTYGG YTTTATYGTV TGRGCYCAYC ACATRTTYAC AGTBGGRATRGAYGTDGAYA CMCGWGCHTA CTTYACATCY GCAACBATAA TYATYGCYAT YCCRACAGGYGTWAAAGTYT TYAGYTGAYT AGCAACYYTV CAYGGRGGCT CARTTAARTG RGAVACHCCBMTMCTBTGRG CCCTDGGYTT YATYTTYCTM TTYACMGTHG GVGGMYTWAC AGGNATYRTHYTRGCYAAYT CYTCYCTAGA YATYGTDCTY CAYGAYACRT AYTAMGTAGT MGCYCAYTTY CA

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T, said sequence SEQ ID N^(o) 58 comprising 442 base pairs, orthe following SEQ ID N^(o)59: GGRYAYATRG GHATRGTNTG AGCYATRATRGCYATYGGMC TYCTYGGYTT TATYGTVTGR GCYCAYCACA TRTTYACAGT BGGRATRGAYGTDGAYACMC GWGCHTACTT YACATCYGCA ACBATAATYA TYGCYATYCC RACAGGYGTWAAAGTYTTYA GYTGAYTAGC ACYYTVCAYG GRGGCTCART TAARTGRGAV ACHCCBMTMCTBTGRGCCCT DGGYTTYATY TTYCTMTTYA CMGTHGGVGG MYTWACAGGN ATYRTHYTRGCYAAYTCYTC YCTAGAYATY GTDCTYCAYG AYACRTAYTA MGTAGTMGCY CAYTTYCA

in which R is A or G, Y is C or T, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T, said sequence SEQ ID N^(o) 59 comprising 328 base pairs, orthe following SEQ ID N^(o)60: AYCARCAYYT RTTCTGATTC TKCGGNCAYCCYGAAGTHTA YATHCTNATY YTMCCHGGMT TCGGRATAAT YTCYCAYATY GTAGCVTAYTAYTCAGGNAA RMAAGARCCN TTYGGRYAYA TRGGHATRGT NTGAGCYATR ATRGCYATYGGMCTYCTYGG YTTTATYGTV TGRGCYCAYC ACATRTTYAC AGTBGGRATR GAYGTDGAYACMCGWGCHTA CTTYACATCY GCAACBATAA TYATYGCYAT YCCRACAGGY GTWAAAGTYTTYAGYTGAYT AGCAACYYTV CAYGGRGGCT CARTTAARTG RGAVACHCCB MTMCTBTGRGCCCTDGGYTT YATYTTYCTM TTYACMGTHG GVGGMYTWAC AGGNATYRTH YTRGCY

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T, said sequence SEQ ID N^(o)60 comprising 386 base pairs, orthe following SEQ ID N^(o)61: GGMCTYCTYG GYTTTATYGT VTGRGCYCAYCACATRTTYA CAGTBGGRAT RGAYGTDGAY ACMCGWGCHT ACTTYACATC YGCAACBATAATYATYGCYA TYCCRACAGG YGTWAAAGTY TTYAGYTGAY TAGCAACYYT VCAYGGRGGCTCARTTAART GRGAVACHCC BMTMCTBTGR GCCCTDGGYT TYATYTTYCT MTTYACMGTHGGVGGMYTWA CAGGNATYRT HYTRGCY

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T, said sequence SEQ ID N^(o) 61 comprising 237 base pairs, orthe following SEQ ID N^(o)62: TAATYTCYCA YATYGTAGCV TAYTAYTCAGGNAARMAAGA RCCNTTYGGR YAYATRGGHA TRGTNTGAGC YATRATRGCY ATYGGMCTYCTYGGYTTTAT YGTVTGRGCY CAYCACATRT TYACAGTBGG RATRGAYGTD GAYACMCGWGCHTACTTYAC ATCYGCAACB ATAATYATYG CYATYCCRAC AGGYGTWAAA GTYTTYAGYTGAYTAGCAAC YYTVCAYGGR GGCTCARTTA ARTGRGAVAC HCCBMTMCTB TGRGCCCTDGGYTTYATYTT YCTMTTYACM GTHGGVGGMY TWACAGGNAT YRTHYTRG

in which Y is C or T, R is A or G, K is G or T, N is A, C, G or T, H isA, C or T, M is A or C, V is A, C or G, B is C, G or T, D is A, G or T,W is A or T, said sequence SEQ ID N^(o) 62 comprising 318 base pairs, orthe following SEQ ID N^(o)63: GRATRGAYGT DGAYACMCGW GCHTACTTYACATCYGCAAC BATAATYATY GCYATYCCRA CAGGYGTWAA AGTYTTYAGY TGAYTAGCAACYYTVCAYGG RGGCTCARTT AARTGRGAVA CHCCBMTMCT BTGRGCCCTD GGYTTYATYTTYCTMTTYAC MGTHGGVGGM YTWACAGGNA TYRTHYTRG

in which R is A or G, Y is C or T, D is A, G or T, M is A or C, W is Aor T, B is C, G or T, V is A, C or G, H is A, C or T, N is A, C, G or T,said sequence SEQ ID N^(o)63 comprising 189 base pairs, or the followingSEQ ID N^(o)64: TYAGYTGAYT AGCAACYYTV CAYGGRGGCT CARTTAARTG RGAVACHCCBMTMCTBTGRG CCCTDGGYTT YATYTTYCTM TTYACMGTHG GVGGMYTWAC AGGNATYRTHYTRGCYAAYT CYTCYCTAGA YATYGTDCTY CAYGAYACRT AYTAMGTAGT MGCYCAYT

in which Y is C or T, V is A, C or G, R is A or G, B is C, G or T, H isA, C or T, M is A or C, D is A, G or T, N is A, C, G or T, said sequenceSEQ ID N^(o)64 comprising 168 base pairs, or the following SEQ IDN^(o)65: CNTTYGGRYA YATRGGHATR GTNTGAGCYA TRATRGCYAT YGGMCTYCTYGGYTTTATYG TVTGRGCYCA YCACATRTTY ACAGTBGGRA TRGAYGTDGA YACMCGWGCHTACTTYACAT CYGCAACBAT AATYATYGCY ATYCCRACAG GYGTWAAAG

in which N is A, C, G or T, R is A or G, Y is C or T, H is A, C or T, Mis A or C, V is A, C or G, B is C, G or T, D is A, G or T, W is A or T,said sequence comprising 159 base pairs, or the following sequence SEQID N^(o)66: TRATRGCYAT YGGMCTYCTY GGYTTTATYG TVTGRGCYCA YCACATRTTYACAGTBGGRA TRGAYGTDGA YACMCGWGCH TACTTYACAT CYGCAACBAT AATYATYGCYATYCCRACAG GYGTWAAAGT YTTYAGYTGA YTAGCAACYY TVCAYGGRGG CTCARTTAARTGRGAVACHC CBMTMCTBTG RGCCCTDG

in which R is A or G, Y is C or T, M is A or C, V is A, C or G, B is C,G or T, D is A, G or T, W is A or T, H is A, C or T. said SEQ ID N^(o)66comprising 198 base pairs, the following SEQ ID N^(o)67: TAATYTCYCAYATYGTAGCV TAYTAYTCAG GNAARMAAGA RCCNTTYGGR YAYATRGGHA TRGTNTGAGCYATRATRGCY ATYGGMCTYC TYGGYTTTAT YGTVTGRGCY CAYCACATRT TYACAGTBGGRATRGAYGTD GAYACMCGWG CHTACTTYAC AT

in which Y is C or T, V is A, C or G, N is A, C, G or T, R is A or G, Mis A or C, H is A, C or T, B is C, G or T, D is A, G or T, W is A or T,said sequence SEQ ID N^(o)67 comprising 162 base pairs, or the followingSEQ ID N^(o)68: GNCAYCCYGA AGTHTAYATH CTNATYYTMC CHGGMTTCGG RATAATYTCYCAYATYGTAG CVTAYTAYTC AGGNAARMAA GARCCNTTYG GRYAYATRGG HATRGTNTGAGCYATRATRG CYATYGGMCT YCTYGGYTTT ATYGTVTGRG CYCAYCACAT RTTYA

in which N is A, C, G or T, Y is C or T, H is A, C or T, M is A or C, Ris A or G, V is A, C or G, said sequence SEQ ID NO 68 comprising 165base pairs.
 14. Method according to claim 1, characterized in that thepresence of mitochondrial DNA originating from gadiformes in a sample oforganic material is detected by amplification of at least one sequenceof mitochondrial DNA specific to the genome of the gadiformes using anyone of the oligonucleotides SEQ ID N^(o)1, SEQ ID N^(o)2, SEQ ID N^(o)3,SEQ ID N^(o)4, SEQ ID N^(o)5, SEQ ID N^(o)6, and any one of theoligonucleotides SEQ ID N^(o)7, SEQ ID N^(o)8, SEQ ID N^(o)9 as definedin claim 6, and advantageously using the pair of oligonucleotides (SEQID N^(o)2 and SEQ ID N^(o)8) or the pair of oligonucleotides (SEQ IDN^(o)5 and SEQ ID N^(o)8), in order to obtain respectively at least oneof the DNA sequences contained in SEQ ID N^(o)58 or in SEQ ID N^(o)59 asdefined in claim 13, said sequences being specific to the genome of thegadiformes, and in that at least one species of gadiforme present insaid sample of organic material is identified by sequencing of at leastone of the DNA sequences contained in SEQ ID N^(o)58 or in SEQ IDN^(o)59.
 15. Method according to any claim 1, characterized in that thepresence of mitochondrial DNA originating from gadidae in a sample oforganic material is detected by amplification of at least one sequenceof mitochondrial DNA specific to the genome of the gadidae using any oneof the oligonucleotides SEQ ID N^(o)10, SEQ ID N^(o)11, SEQ ID N^(o)12,SEQ ID N^(o)13, SEQ ID N^(o)14, SEQ ID N^(o)15, and any one of theoligonucleotides SEQ ID N^(o)16, SEQ ID N^(o)17, SEQ ID N^(o)18 asdefined in claim 6, and advantageously using the pair ofoligonucleotides (SEQ ID N^(o)11 and SEQ ID N^(o)17) or the pair ofoligonucleotides (SEQ ID N^(o)14 and SEQ ID N^(o)17), in order to obtainrespectively at least one of the DNA sequences contained in SEQ IDN^(o)60 or in SEQ ID N^(o)61 as defined in claim 13, said sequencesbeing specific to the genome of the gadidae, and in that at least onespecies of gadidae present in said sample of organic material isidentified by sequencing of at least one of the DNA sequences containedin SEQ ID N^(o)60 or in SEQ ID N^(o)61.
 16. Method according to claim 1,characterized in that the presence of DNA originating from merluccidaein a sample of organic material is detected by amplification of at leastone sequence of mitochondrial DNA specific to the genome of themerluccidae using any one of the oligonucleotides SEQ ID N^(o)19, SEQ IDN^(o)20, SEQ ID N^(o)21, SEQ ID N^(o)22, SEQ ID N^(o)23, SEQ ID N^(o)24,and any one of the oligonucleotides SEQ ID N^(o)25, SEQ ID N^(o)26, SEQID N^(o)27 as defined in claim 6, and advantageously using the pair ofoligonucleotides (SEQ ID N^(o)20 and SEQ ID N^(o)26) or the pair ofoligonucleotides (SEQ ID N^(o)23 and SEQ ID N^(o)26), in order to obtainrespectively at least one of the DNA sequences contained in SEQ IDN^(o)62 or in SEQ ID N^(o)63 as defined in claim 13, said sequencesbeing specific to the genome of the merluccidae, and in that at leastone species of merluccidae present in said sample of organic material isidentified by sequencing of at least one of the DNA sequences containedin SEQ ID N^(o)62 or in SEQ ID N^(o)63.
 17. Method according to claim 1,characterized in that the presence of mitochondrial DNA originating fromgadiformes is identified in a sample of organic material, and inparticular of gadidae chosen from the group constituted by the speciesGadus morhua (common cod), Pollachius virens (pollock), Theragrachalcogramma (Alaskan pollock), Melanogrammus aeglefinus (haddock) andMerlangius merlangus (whiting), and in that each of the aforementionedspecies is identified by amplification of at least one DNA sequencespecific to the genome of each of the aforementioned species of gadidae,with the help respectively: of any one of the oligonucleotides SEQ IDN^(o)28, SEQ ID N^(o)29, SEQ ID N^(o)30, and any one of theoligonucleotides SEQ ID N^(o)31, SEQ ID N^(o)32, SEQ ID N^(o)33 asdefined in claim 6 or, of any one of the oligonucleotides SEQ IDN^(o)34, SEQ ID N^(o)35, SEQ ID N^(o)36, and any one of theoligonucleotides SEQ ID N^(o)37, SEQ ID N^(o)38, SEQ ID N^(o)39 asdefined in claim 6 or, of any one of the oligonucleotides SEQ IDN^(o)40, SEQ ID N^(o)41, SEQ ID N^(o)42, and any one of theoligonucleotides SEQ ID N^(o)43, SEQ ID N^(o)44, SEQ ID N^(o)45 asdefined in claim 6 or, of any one of the oligonucleotides SEQ IDN^(o)46, SEQ ID N^(o)47, SEQ ID N^(o)48, and any one of theoligonucleotides SEQ ID N^(o)49, SEQ ID N^(o)50, SEQ ID N^(o)51 asdefined in claim 6 or, of any one of the oligonucleotides SEQ IDN^(o)52, SEQ ID N^(o)53, SEQ ID N^(o)54, and any one of theoligonucleotides SEQ ID N^(o)55, SEQ ID N^(o)56, SEQ ID N^(o)57 asdefined in claim 6, and advantageously using respectively: the pair ofoligonucleotides (SEQ ID N^(o)29 and SEQ ID N^(o)32) or, the pair ofoligonucleotides (SEQ ID N^(o)35 and SEQ ID N^(o)38) or, the pair ofoligonucleotides (SEQ ID N^(o)41 and SEQ ID N^(o)44) or, the pair ofoligonucleotides (SEQ ID N^(o)47 and SEQ ID N^(o)50) or, the pair ofoligonucleotides (SEQ ID N^(o)53 and SEQ ID N^(o)56), in order to obtainrespectively at least one of the DNA sequences contained in: SEQ IDN^(o)64 specific to the genome of Gadus morhua (common cod), SEQ IDN^(o)65 specific to the genome of Pollachius virens (pollock), SEQ IDN^(o)66 specific to the genome of Theragra chalcogramma (Alaskanpollock), SEQ ID N^(o)67 specific to the genome of Melanogrammusaeglefinus (haddock), SEQ ID N^(o)68 specific to the genome ofMerlangius merlangus (whiting), said SEQ ID N^(o)64, SEQ ID N^(o)65, SEQID N^(o)66, SEQ ID N^(o)67 and SEQ ID N^(o)68.
 18. A method fordetecting the presence of biological materials originating fromgadiformes, comprising screening for nucleotide sequences chosen fromthe oligonucleotide primers according to claim 6 or from sequences CGGGAT CCT GTT CTG ATT CTT GAT TTC C (SEQ ID NO: 69) and CGA CGG GAT CCCAAC ACC TGT TTC GAT CAT CGC GGC AAC (SEQ ID NO: 70).